antibodies binding to a C-terminal fragment of apoliopoprotein E

ABSTRACT

A human antibody fragment, which antibody or fragment: (i) binds to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 of the C-terminal domain of Apolipoprotein E (ApoE-CTD) or the amino acid sequence of a part thereof; and (ii) binds to human plaques.

This application is a U.S. national stage of International PatentApplication No. PCT/EP2004/013426, filed 26 Nov. 2004, which designatedthe U.S. and claims priority of U.S. Provisional Application No.60/525,174, filed 28 Nov. 2003; the entire contents of each of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to antibodies that specifically bind to aC-terminal fragment of Apolipoprotein E (ApoE). The present inventionalso provides methods for obtaining such polypeptides and the use ofsuch polypeptides in the diagnosis and treatment of Alzheimer's disease,systemic amyloidosis and other amyloid disorders.

BACKGROUND TO THE INVENTION

Amyloidosis is a progressive, incurable metabolic disease of unknowncause characterized by abnormal deposits of protein in one or moreorgans or body systems. Amyloid proteins are manufactured, for example,by malfunctioning bone marrow. Amyloidosis, which occurs whenaccumulated amyloid deposits impair normal body function, can causeorgan failure or death. It is a rare disease, occurring in about eightof every 1,000,000 people. It affects males and females equally andusually develops after the age of 40. At least 15 types of amyloidosishave been identified. Each one is associated with deposits of adifferent kind of protein.

The major forms of amyloidosis are primary systemic, secondary, andfamilial or hereditary amyloidosis. There is also another form ofamyloidosis associated with Alzheimer's disease. Primary systemicamyloidosis usually develops between the ages of 50 and 60. With about2,000 new cases diagnosed annually, primary systemic amyloidosis is themost common form of this disease in the United States. Also known aslight-chain-related amyloidosis, it may also occur in association withmultiple myeloma (bone marrow cancer). Secondary amyloidosis is a resultof chronic infection or inflammatory disease. It is often associatedwith Familial Mediterranean fever (a bacterial infection characterizedby chills, weakness, headache, and recurring fever), Granulomatousileitis (inflammation of the small intestine), Hodgkin's disease,Leprosy, Osteomyelitis and Rheumatoid arthritis.

Familial or hereditary amyloidosis is the only inherited form of thedisease. It occurs in members of most ethnic groups, and each family hasa distinctive pattern of symptoms and organ involvement. Hereditaryamyloidosis is though to be autosomal dominant, which means that onlyone copy of the defective gene is necessary to cause the disease. Achild of a parent with familial amyloidosis has a 50-50 chance ofdeveloping the disease.

Amyloidosis can involve any organ or system in the body. The heart,kidneys, gastrointestinal system, and nervous system are affected mostoften. Other common sites of amyloid accumulation include the brain,joints, liver, spleen, pancreas, respiratory system, and skin.

Alzheimer's disease (AD) is the most common form of dementia, aneurologic disease characterized by loss of mental ability severe enoughto interfere with normal activities of daily living, lasting at leastsix months, and not present from birth. AD usually occurs in old age,and is marked by a decline in cognitive functions such as remembering,reasoning, and planning.

Between two and four million Americans have AD; that number is expectedto grow to as many as 14 million by the middle of the 21st century asthe population as a whole ages. While a small number of people in their40s and 50s develop the disease, AD predominantly affects the elderly.AD affects about 3% of all people between ages 65 and 74, about 20% ofthose between 75 and 84, and about 50% of those over 85. Slightly morewomen than men are affected with AD, even when considering women tend tolive longer, and so there is a higher proportion of women in the mostaffected age groups.

Several genes have been implicated in AD, including the gene for amyloidprecursor protein, or APP, responsible for producing amyloid. Mutationsin this gene are linked to some cases of the relatively uncommonearly-onset forms of AD. Other cases of early-onset AD are caused bymutations in the presenilin genes, PS-1 and PS-2. A dementia similar toAD eventually affects nearly everyone with Downs syndrome, caused by anextra copy of chromosome 21. Other mutations on other chromosomes havebeen linked to other early-onset cases.

Potentially the most important genetic link was discovered in the early1990s on chromosome 19. A gene on this chromosome, apoE, codes for aprotein involved in transporting lipids into neurons.

Apolipoprotein E (ApoE) is a 34 kDa glycosylated protein. The main sitesof ApoE production are the liver and brain. ApoE is a constituent ofvery low density lipoprotein (VLDL), a subclass of high densitylipoproteins and chylomicrons. Cellular uptake of lipid complexes ismediated by binding of ApoE to the low density lipoprotein (LDL)receptor and other related receptors.

There are three major ApoE isoforms in humans, apoE2, apoE3 and apoE4which are products of three alleles, ε2, ε3 and ε4. In the generalpopulation, the ε3 allele is the most common, accounting for 78% of allapoE alleles. The frequency of the ε4 allele is increased significantlyin the population of late-onset sporadic and familial Alzheimer'sdisease (AD) patients.

ApoE contains a C-terminal domain (ApoE-CTD) and an N-terminal domain(ApoE-NTD) joined by a random-coil region. The C-terminal domaincomprises a lipid binding site and the N-terminal domain binds tolipoprotein receptors. The CTD amino acid sequence is identical in allthree isoforms of ApoE. The CTD and NTD may be separated by cleavagewith thrombin.

Direct interactions between ApoE and Amyloid β(Aβ) have beendemonstrated in vitro. ApoE is also present in AD plaques. It has beenreported that the N-terminal domain of ApoE (ApoE-NTD) mediates bindingof apoE to Aβ(Golabek et al., (2000) Biophysical Journal 79: 1008-1015).However, AD plaques containing ApoE have been shown to comprisefull-length ApoE at the centre of the plaques and a C-terminal domainfragment of ApoE (ApoE-CTD) at the periphery of the plaques (Cho et al.,(2001) J. Neuropathology and Expt. Neurology 60: 342-349). Aβ₁₋₄₂deposition in plaques has been shown to precede ApoE deposition whilstAβ₁₋₄₀ deposition follows ApoE depositon in plaque maturation (Terai etal., (2001), Brain Research 900: 48-56).

The function of ApoE in the brain is not thought to be specific for AD.ApoE appears to play an important role in modifying recovery from acutebrain injury. In particular, there is evidence from both clinical andanimal studies to suggest that the presence of the ApoE4 isoform isassociated with poor neurological recovery from a variety of acute braininjuries.

SUMMARY OF THE INVENTION

The present inventors developed therapeutic antibodies directed to aregion of Apolipoprotein E (ApoE) which is exposed in protein aggregatesfound in amyloid deposits including Alzheimer plaques but which is notaccessible, or has only restricted accessibility, in other forms of ApoEsuch as ApoE in lipoprotein particles in the blood.

Accordingly, the present invention provides:

-   -   a human antibody or antibody fragment, which antibody or        fragment:

(i) binds to a polypeptide having the amino acid sequence shown in SEQID NO: 1 of the C-terminal domain of Apolipoprotein E (ApoE-CTD) or theamino acid sequence of a part thereof; and

(ii) binds to human plaques;

-   -   a human antibody or antibody fragment, which antibody or        fragment:

(i) binds to a polypeptide having the amino acid sequence shown in SEQID NO: 1 of ApoE-CTD or the amino acid sequence of a part thereof; and

(ii) comprises a heavy chain CDR3 region comprising the sequence shownin SEQ ID NO: 20, SEQ ID NO: 512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ IDNO: 515, SEQ ID NO: 516 or SEQ ID NO: 517;

-   -   a human antibody or antibody fragment, which antibody or        fragment:

(i) binds to a polypeptide having the amino acid sequence shown in SEQID NO: 1 of ApoE-CTD or the amino acid sequence of a part thereof; and

(ii) comprises a heavy chain CDR3 region comprising an amino acidsequence selected from the sequences shown in SEQ ID NO: 29, SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ IDNO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 74, SEQID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 86 and SEQ ID NO:89;

-   -   a human antibody or antibody fragment, which antibody or        fragment binds, in the presence of VLDL, to a polypeptide having        the ApoE-CTD amino acid sequence shown in SEQ ID NO: 1 or the        amino sequence of a part thereof;    -   a human antibody or antibody fragment, which antibody or        fragment:

(i) binds to human plaques; and

(ii) comprises a heavy chain CDR3 region comprising the sequence shownin SEQ ID NO: 20, SEQ ID NO: 512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ IDNO: 515, SEQ ID NO: 516 or SEQ ID NO: 517;

-   -   a human antibody or antibody fragment, which antibody or        fragment:

(i) binds to human plaques; and

(ii) comprises a heavy chain CDR3 region comprising an amino acidsequence selected from the sequences shown in SEQ ID NO: 29, SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ IDNO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 74, SEQID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 86 and SEQ ID NO:89;

-   -   an antibody or antibody fragment which comprises the heavy chain        sequence shown in SEQ ID NO: 136 and the light chain sequence        shown in SEQ ID NOS: 521 and 522;    -   an antibody or antibody fragment which comprises the heavy chain        sequence shown in SEQ ID NO: 142 and the light chain sequence        shown in SEQ ID NO: 523;    -   an antibody or antibody fragment which comprises the heavy chain        sequence shown in SEQ ID NO: 40 and the light chain sequence        shown in SEQ ID NO: 517 and/or 518;    -   an antibody or antibody fragment which comprises the heavy chain        sequence shown in SEQ ID NO: 40 and the light chain sequence        shown in SEQ ID NO: 519 and/or 520;    -   an antibody or antibody fragment which comprises the heavy chain        CDR1 sequence shown in SEQ ID NO: 24, the heavy chain CDR2        sequence shown in SEQ ID NO: 25 and the heavy chain CDR3        sequence shown in any one of SEQ ID NOS: 207, 209 and 210;    -   an antibody or antibody fragment which comprises the heavy chain        CDR1 sequence shown in SEQ ID NO: 48, the heavy chain CDR2        sequence shown in SEQ ID NO: 49 and the heavy chain CDR3        sequence shown in any one of SEQ ID NOS: 320, 322 and 323;    -   an antibody or antibody fragment which comprises the heavy chain        CDR1 sequence shown in SEQ ID NO: 66, the heavy chain CDR2        sequence shown in SEQ ID NO: 67 and the heavy chain CDR3        sequence shown in SEQ ID NO: 373;    -   an antibody or antibody fragment according to any one of the        preceding claims which is a monoclonal antibody,    -   an antibody or antibody fragment according to the invention, for        use in a method of treatment of the human or animal body by        therapy or in a diagnostic method practiced on the human or        animal body,    -   use of an antibody or antibody fragment according to the        invention, in the manufacture of a medicament for the treatment        or prevention of an amyloid disorder;    -   a pharmaceutical composition comprising an antibody or antibody        fragment according to the invention and a pharmaceutically        acceptable carrier or diluent;    -   a method of treating a subject suffering from an amyloid        disorder comprising administering to said subject a        therapeutically effective amount of an antibody or antibody        fragment according to the invention;    -   a method of diagnosing an amyloid disorder in a subject        comprising:

(i) administering to said subject an antibody or antibody fragmentaccording to the invention; and

(ii) determining whether or not said antibody or antibody fragment bindsto plaques in said subject, wherein binding of said antibody or antibodyfragment to plaques is indicative of an amyloid disorder, therebydetermining whether the subject has an amyloid disorder;

-   -   a polynucleotide encoding an antibody or antibody fragment        according to the invention;    -   a vector comprising a polynucleotide according to the invention;    -   a host cell expressing a polypeptide according the invention;    -   a virus encoding a polynucleotide according to the invention;    -   a kit for detecting ApoE-CTD, which kit comprises an antibody or        antibody fragment according to the invention and means for        detecting said an antibody or antibody fragment; and

a method for detecting the presence of ApoE-CTD in a sample from asubject, which method comprises:

(i) contacting a sample taken from a subject with an antibody orantibody fragment according to the invention under conditions thatpermit binding of the an antibody or antibody fragment to ApoE-CTD; and

(ii) determining whether or not the an antibody or antibody fragmentbinds to the sample thereby detecting any ApoE-CTD present in thesample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the binding of known monoclonal antibodies to biotinylatedCTD (bCTD) (A) and VLDL (B) by ELISA.

FIG. 2 shows the binding of phage to bCTD (background=0.05) (A) and VLDL(background=0.1) (B).

FIG. 3 shows an example of bCTD (A) and VLDL (B) ELISA on 203 phageclones.

FIG. 4 shows Biacore affinity analysis on bCTD coated of sFab antibodiesM27E11 (A), M28B02 (B) and M26 F05 (C) and Biacore affinity analysiswith Fab M27E11 coated on the chip (D).

FIG. 5 is a schematic diagram showing the strategy of transfer of Fab topBh1.

FIG. 6 shows the binding of antibodies 807A-M0028-B02 (M28B02) (A),807A-M0026-F05 (M26F05) (B) and 807A-M0027-E11 (M27E11) (C) to humanCTD, murine CTD and primate CTD.

FIG. 7 shows the binding of control antibody PH1 (A) and 807A-M0028-B02(M28B02) (B) to bCTD and bNTD.

FIG. 8 shows the binding of control antibody PH1 (A) and 807A-M0028-B02(M28B02) (B) to coated VLDL.

FIG. 9 shows the results of antibody binding in competition ELISAbetween coated bCTD (0.05 μg/mI) and an excess of VLDL or CTD insolution. Binding of M27E11 IgG (A), M28B2 IgG (B), control antibody (C)and PH1 IgG (D) is shown.

FIG. 10 shows the Biacore analysis of 807A-M0026-F05 (M26F05) as solubleFab (top) and IgG (bottom) on a CTD-control chip.

FIG. 11 shows the Biacore analysis of 807A-M0027-E11 (A) and807A-M0028-B02 (B) as soluble Fab (top) and IgG (bottom) on a CTD-coatedchip.

FIGS. 12 and 13 show the binding of bCTD in solution to Fab807A-M0027-E1 (M27E11) indirectly coupled to a Biacore chip.

FIG. 14 is a schematic diagram showing the strategy used to transferV-regions from pBh1 to pRmk2a.

FIG. 15 shows the sequences of the CTD peptides.

FIG. 16 shows the selection campaign of Example 21.

FIG. 17 shows the selection campaign of Example 22.

FIG. 18 shows the selection campaign of Example 23.

FIG. 19 shows a ‘cut and paste’ antibody reformatting strategy.

FIG. 20 shows that 807A-M0028-B02 plaque binding in human AD brainsections is not blocked in presence of VLDL. Binding of 807A-M0028-B02to human amyloid plaques in vitro was not affected by the presence ofVLDL indicating that the antibody has very low affinity for CTD in VLDLrelative to CTD in plaques.

FIG. 21 shows the results of an in vitro immunohistochemistry (IHC)screen of sFab antibody clones on human AD brain sections. Severalantibodies (Fab clones) from different selections were identified thatbind to AD plaques by immunohistochemistry. Note that individual clonesmay have the same short name i.e. E11 but are from different selectionsand not identical. (C11=807B-M0004-H03; (selection A)E11=807A-M0027-E11; B2=807A-M0028-B02; (selection B) E11=807B-M0083-E11;D10=807B-M0079D10; A3=807B-M0004-A03; A12=807B-M0013-A12)

FIG. 22 shows representative results (duplicate samples) of theconcentration dependent binding of 807A-M0028-B02, 807A-M0028-B02.1 and807A-M0028-B02.2 to human, primate and murine ApoE-CTD.

FIG. 23 shows representative results (duplicate samples) of theconcentration dependent binding of 807A-M0028-B02, 807A-M0028-B02.1,807A-M0028-B02.2, 807B-M0004-H03, 807B-M0004-H03.1, 807B-M0004-A03 and807B-M0004-A03.1 to human VLDL.

FIG. 24 shows the detection of 807A-M0028-B02 plaque binding in braintissue sections from APP/PS1 mouse:

a) In vivo binding of 807A-M0028-B024 days after injection;

b) In vivo binding of 807A-M0028-B027 days after injection.

FIG. 25 shows results of a screen of in vivo plaque binding capacity of807B M0004H03, 807B-M04-A03, 807B-M0079-D10 and 807B-M0009-F06.Immunohistochemistry expression patterns of anti-CTD hIgG clonesB807B-M0004H03, 807B-M0004-A03, 807B-M0079-D10 and 807B-M0009-F06 inAPP/PS1 mouse brain sections after in vivo administration is shown.

FIG. 26 shows results of an in vitro screen for plaque binding capacityof affinity maturated Fab-clones in human AD brain sections. Wild-typeclone 807B-M0004-A03 (wt A03) was compared to affinity maturated clones807B-M 0118-B09 (BO9), 807B-M 0117-F05 (F05), 807B-M 0117-G01 (G01) and807B-M 0118-F03 (F03). Amyloid plaques visualized by anti-CTD bindingantibodies (Fab clones) on human AD brain sections.

FIG. 27 shows the strategy used for affinity maturation of807A-M0028-B02, 807B-M0004-H03, 807B-M004-A03, 807B-M0079-D10 and807B-M0009-F06.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of the ApoE-CTD.

SEQ ID NO: 2 is the amino acid sequence of peptide 1 (amino acids 1 to16 of the ApoE-CTD).

SEQ ID NO: 3 is the amino acid sequence of peptide 2 (amino acids 17 to32 of the ApoE-CTD).

SEQ ID NO: 4 is the amino acid sequence of peptide 3 (amino acids 33 to48 of the ApoE-CTD).

SEQ ID NO: 5 is the amino acid sequence of peptide 4 (amino acids 49 to64 of the ApoE-CTD).

SEQ ID NO: 6 is the amino acid sequence of peptide 5 (amino acids 65 to80 of the ApoE-CTD).

SEQ ID NO: 7 is the amino acid sequence of peptide 6 (amino acids 9 to24 of the ApoE-CTD).

SEQ ID NO: 8 is the amino acid sequence of peptide 7 (amino acids 25 to40 of the ApoE-CTD).

SEQ ID NO: 9 is the amino acid sequence of peptide 8 (amino acids 41 to56 of the ApoE-CTD).

SEQ ID NO: 10 is the amino acid sequence of peptide 9 (amino acids 57 to72 of the ApoE-CTD).

SEQ ID NO: 11 is the amino acid sequence of peptide 10 (amino acids 73to 84 of the ApoE-CTD).

SEQ ID NO: 12 is the amino acid sequence of an epitope in peptide 4(amino acids 53 to 60 of ApoE-CTD).

SEQ ID NO: 13 is the amino acid sequence of an epitope in peptides 4 and9 (amino acids 57 to 64 of ApoE-CTD).

SEQ ID NO: 14 is the amino acid sequence of an epitope in peptide 9(amino acids 61 to 68 of ApoE-CTD).

SEQ ID NO: 15 is the amino acid sequence of an epitope in peptides 1 and6 (amino acids 9 to 16 of ApoE-CTD).

SEQ ID NO: 16 is the amino acid sequence of an epitope in peptides 4 and8 (amino acids 49 to 56 of ApoE-CTD).

SEQ ID NO: 17 is the amino acid sequence of an epitope in peptides 3 and8 (amino acids 41 to 48 of ApoE-CTD).

SEQ ID NO: 18 is the amino acid sequence of peptides 1 and 6 (aminoacids 1 to 24 of ApoE-CTD).

SEQ ID NO: 19 is the amino acid sequence of peptides 8 and 9 (aminoacids 41 to 72 of ApoE-CTD).

SEQ ID NO: 20 is the amino acid sequence of the consensus CDR3 sequencefrom antibodies 807A-M0027-E11 and 807A-M0028-B02.

SEQ ID NOS: 21 to 164 are described in Table 8.

SEQ ID NO: 165 is the amino acid sequence of human ApoE4.

SEQ ID NO: 166 is the amino acid sequence of human ApoE3.

SEQ ID NO: 167 is the amino acid sequence of human ApoE2.

SEQ ID NO: 168 is the amino acid sequence of the mature form of humanApoE4.

SEQ ID NO: 169 is the amino acid sequence of the mature form of humanApoE3.

SEQ ID NO: 170 is the amino acid sequence of the mature form of humanApoE2.

SEQ ID NOS: 171 to 206 are described in Table 8.

SEQ ID NOS: 207 to 511 are described in Tables 38 to 42.

SEQ ID NO: 512 is the consensus amino acid sequence of the CDR3 regionsof affinity matured clones of 807A-M0028-B02.

SEQ ID NO: 513 is the consensus amino acid sequence of the CDR3 regionsof affinity matured clones of 807B-M0004-A03.

SEQ ID NO: 514 is the consensus amino acid sequence of the CDR3 regionsof affinity matured clones of 807B-M0004-H03.

SEQ ID NO: 515 is the consensus amino acid sequence of the CDR3 regionsof affinity matured clones of 807B-M0009-F06.

SEQ ID NO: 516 is the consensus amino acid sequence of the CDR3 regionsof selected affinity matured clones of 807A-M0028-B02.

SEQ ID NO: 517 is the consensus amino acid sequence of the CDR3 regionsof selected affinity matured clones of 807B-M0004-A03.

SEQ ID NO: 518 to 527 are defined in Table 21.

DETAILED DESCRIPTION OF THE INVENTION

A. Polypeptides

The present invention provides antibodies that bind to a region onApolipoprotein E (ApoE) which is exposed in the protein aggregates foundin amyloid deposits such as Alzheimer plaques, but which is not presentor accessible in other forms of ApoE, such as in lipoprotein particlesin the blood.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes antibody fragments.

Typically, an antibody of the invention binds the C-terminal domain ofApolipoprotein E (ApoE-CTD), i.e. is reactive with ApoE-CTD. An antibodyof the invention does not bind to the N-terminal domain ofApolipoprotein E (ApoE-NTD). The antibody typically binds to the form ofApoE present in human plaques in preference to the form of ApoE presentin VLDL. Generally, the form of ApoE present in human plaques isApoE-CTD. An antibody of the invention preferably binds ApoE-CTD in thepresence of very low density lipoprotein (VLDL). An antibody of theinvention may be one that binds to an epitope in ApoE-CTD, which epitopeis not present in ApoE associated with VLDL. For example, the epitopemay be one which is one not accessible or exposed to the antibody whenApoE is associated with VLDL. The epitope to which the antibody bindsmay typically be hidden in full-length ApoE present in VLDL and so theaffinity of the antibody for ApoE is substantially less than itsaffinity for ApoE-CTD. The epitope to which the antibody binds ispresent only in ApoE-CTD and not in ApoE-NTD and so the antibody istypically devoid of binding to ApoE-NTD. Any binding of the antibody toApoE-NTD is generally non-specific binding of a substantially loweraffinity than the specific binding of the antibody to ApoE-CTD. Asubstantially lower affinity is generally at least a two fold, threefold, five fold, 10 fold, 50 fold or 100 fold lower affinity.

An antibody of the invention thus preferentially binds or specificallybinds to ApoE-CTD. An antibody “preferentially binds” or “specificallybinds” to ApoE-CTD when it binds with preferential or high affinity toApoE-CTD but does not substantially bind, does not bind or binds withonly low affinity to other polypeptides. A variety of protocols forbinding, competitive binding or immuno-radiometric assays to determinethe specific binding capability of an antibody are well known in the art(see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993). Suchimmunoassays typically involve the formation of complexes between thespecific protein and its antibody and the measurement of complexformation. Typically an antibody of the invention, is capable of bindingto ApoE-CTD having the sequence shown in SEQ ID NO: 1 with an affinityconstant of at least 10⁷ M⁻¹, preferably at least 10⁸ M⁻¹, 10⁹ M⁻¹ or10¹⁰ M⁻¹. An antibody of the invention, is preferably capable ofpreferentially binding to ApoE-CTD with an affinity that is at leasttwo-fold, 10-fold, 50-fold, 100-fold or greater than its affinity forbinding to a non-specific polypeptide such as BSA, casein, VLDL,ApoE-NTD or ApoE present in VLDL.

An antibody which specifically binds to ApoE-CTD typically displays atleast 2× background binding in an ELISA on immobilised ApoE-CTD but lessthan 2× background, typically 1× background, to control proteins such asApoE-NTD or streptavidin.

An antibody of the invention generally binds to human plaques. The term“human plaques” is intended to cover any amyloid deposits comprising atleast one protein having an amino acid sequence encoded by a human gene.Preferably the human plaque is present in or derived from human tissue.More preferably the human plaque is present in a sample that has beenobtained from a human subject. The human subject may have an amyloiddisorder, such as systemic amyloidosis or Alzheimer's disease. Thesample may be taken from any tissue or organ containing amyloid plaques.Suitable tissues and organs include brain, tongue, intestines, skeletalmuscle, smooth muscle, nerves, skin, ligaments, heart, liver, spleen andkidneys. Where the subject has Alzheimer's disease, the sample isgenerally a brain section. The brain section is typically obtainedpost-mortem. Fibrils prepared from any such sample are also includedwithin the term “human plaques”.

The human plaque may be present in or derived form a non-human animalwhich is transgenic for one or more, for example two or three, humanproteins, which human protein(s) is/are found in amyloid deposits. Thehuman protein is preferably ApoE but may be amyloid precursor protein(APP) (typically comprising the Swedish mutation) or presenilin.

Binding to human plaques may be determined by any suitable method. Forexample in an IHC assay, binding of an antibody to human plaques can besaid to occur when a positive blind scored IHC signal is obtained afterstaining with <20 μg/ml antibody in two amyloid deposit samplesprimarily tested or if one sample is negative in the primary test, atleast two out of three samples subsequently tested indicates that anantibody binds to human plaques. The samples are preferably derived fromdifferent individuals and sectioned from tissue samples withhistologically verified amyloid deposits which are IHC positive for anamyloid marker such as Aβ.

The ability of an antibody to bind to human plaques may be determined invivo using a mouse or other non-human animal model, such as a rodent orprimate, of Alzheimer's disease or systemic amyloidosis.

In such an assay, binding of an antibody to plaques may be determinedusing IHC. The antibody may be labelled prior to being tested. Bindingto the plaque may be defined as positive blind scored IHC staining ofamyloid after injection of ≦1 mg antibody, in single or multiple doses,in at least two out of three mice tested. The signal is generallycompared to the signal from stained anatomically, sex and age matchedtissue from negative isotype matched control antibody injected mice.

The term “epitope” as used herein refers to that portion of a moleculethat makes contact with a particular binding polypeptide. An epitope maybe linear, comprising an essentially linear amino acid sequence from theantigen or conformational, comprising sequences that are separated byother sequences but come together structurally to form a binding sitefor the polypeptide.

The epitope in ApoE-CTD to which the antibody binds may appear onApoE-CTD after cleavage from full-length ApoE. Alternatively the epitopemay appear following the interaction of ApoE-CTD with amyloid plaques,for example as a result of binding of ApoE-CTD to Aβ. Cleavage of ApoEand/or binding of ApoE-CTD to amyloid plaques may result in the exposureof new linear (peptide) epitopes and/or to the exposure or formation ofnew conformational epitopes. The epitope to which a polypeptide of theinvention binds may be hidden in VLDL-associated ApoE due to theinteraction of ApoE with other components of VLDL. The polypeptide maybind specifically to a complex formed between ApoE-CTD and Aβ.

The amino acid sequence of ApoE-CTD is shown in SEQ ID NO: 1. ApoE-CTDepitopes may thus be formed by a linear or conformational sequencewithin the sequence of ApoE-CTD as shown in SEQ ID NO: 1. An antibodythat binds to ApoE-CTD typically bind to an ApoE-CTD polypeptide havingthe whole sequence shown in SEQ ID NO: 1 but may also bind to a part ofthe amino acid sequence of SEQ ID NO: 1 such as to a peptide having anamino acid sequence as shown in any one of SEQ ID NOS: 2 to 19.Preferably, the antibody binds to one or more of the peptides shown inSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, or SEQ ID NO: 17. The part of ApoE-CTD to which the antibody bindsis at least a three amino acid fragment of SEQ ID NO: 1, preferably atleast a five, six, seven or eight amino acid fragment, more preferably a10, 12 or 16 amino acid fragment.

The polypeptide (or peptide) to which the antibody binds may be arecombinant polypeptide. The polypeptide may be in solution or, morepreferably, may be attached to a solid surface. For example, thepolypeptide may be attached to beads, such as magnetic beads.

The polypeptide may be biotinylated. The biotin molecule conjugated tothe peptide may be used to immobilize the polypeptide on a solid surfaceby coupling biotin to streptavidin on the solid surface.

An antibody of the present invention suitable for use in treating orpreventing Alzheimer's disease and/or systemic amyloidosis typicallytests positive in an ex vivo phagocyte assay. A positive phagocyte assayis defined as positive blind scored confocal microscopy detection ofphagocytes that contain amyloid after co-culture on amyloid tissue afterapplying ≦20 μg/ml of the antibody in at least two out of three culturestested. The signal is generally compared to the signal from identicalco-cultures containing the same concentration of a negative controlantibody. A positive phagocyte assay generally also results in thedegradation of amyloid, for example as shown by Western blot to be lessthan one third the density of the Aβ-based remaining after up to threedays of co-culture, as compared to blots from identical co-culturescontaining the same concentration of a negative-control antibody.

Antibodies and other peptides for therapeutic use are typically of highaffinity, preferably having an affinity of <1 nM, for ApoE-CTD, toenable them to function optimally even at the low concentrations in thebrain that will build up after systemic injection.

The term “antibody” refers to a protein comprising at least one, andpreferably two, heavy chain variable regions (VH) and/or at least one,preferably two, light chain variable regions (VL). The VH and VL regionscan be further subdivided into regions of hypervariability, termed“complementarity determining regions (CDR)”, interspersed with regionsthat are more conserved, termed “framework regions (FR)”. The extent ofthe FR and CDRs has been precisely defined (see, Kabat, et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Chothia et al. (1987) J. Mol. Biol. 196: 901-917, which are incorporatedherein by reference in their entirety). Each VH and VL is composed ofthree CDRs and four FRs arranged from N-terminus to C-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain respectively. In one embodiment, the antibody is atetramer of two heavy and two light chains, wherein the heavy and lightchains are interconnected by, for example, disulphide bonds. The heavychain constant region is comprised of three domains, CH1, CH2 and CH3.The light chain constant region is comprised of one domain, CL. Thevariable region of the heavy and light chains contains a binding domainthat interacts with antigen. The constant regions of the antibodiestypically mediate the binding of the antibody to host tissues andfactors, including various cells of the immune system and the firstcomponent of the complement system. The term “antibody” includes intactimmunoglobulins of types IgA, IgG, IgE, IgD, IgM and subtypes thereof. Apreferred immunoglobulin is IgG.

As used herein the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. The recognised human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu conatant region genes as well as a myriad ofimmunoglobulin variable region genes. Full-length immunoglobulin lightchains (about 25 kD or 214 amino acids) are encoded by a variable regiongene at the N-terminus (about 110 amino acids) and a kappa or lambdaconstant region at the C-terminus. Full-length immunoglobulin heavychains (about 50 kD or 446 amino acids) are encoded by a variable regiongene at the N-terminus (about 116 amino acids) and one of the otheraforementioned constant region genes at the C-terminus, e.g. gamma(encoding about 330 amino acids).

An antibody fragment of the invention is typically an antigen-bindingfragment. The term “antigen-binding fragment” refers to one or morefragments of a full-length antibody that are capable of specificallybinding to ApoE-CTD. Examples of binding fragments include (i) a Fabfragment (a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment (a bivalent fragment comprising two Fabfragments linked by a disulphide bridge at the hinge region; (iii) a Fdfragment (consisting of the VH and CH1 domains); (iv) a Fv fragment(consisting of the VH and VL domains of a single arm of an antibody);(v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR;(vii) a single chain Fv (scFv) (consisting of the VH and VL domains of asingle arm of an antibody joined by a synthetic linker using recombinantmeans such that the VH and VL domains pair to form a monovalentmolecule); (viii) diabodies (consisting of two scFvs in which the VH andVL domains are joined such that they do not pair to form a monovalentmolecule; the VH of each one of the scFv pairs with the VL domain of theother scFv to form a bivalent molecule); (ix) bi-specific antibodies(consisting of at least two antigen binding regions, each region bindinga different epitope). Preferably, the antibody fragment is a Fabfragment or single-chain antibody (scFv).

The sequences of preferred CDR1 domains are shown in SEQ ID NOS: 21, 24,27, 30, 33, 36, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84,87, 93, 111, 117 and 123. Other preferred CDR1 domains are variants ofthese sequences in which one or more amino acids within the sequencehave been deleted or, more preferably, substituted. Other preferred CDR1domains are variants of the sequences shown in any one of SEQ ID NOS:21, 24, 27, 30, 33, 36, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78,81, 84, 87, 93, 111, 117 and 123 in which one or more amino acid hasbeen inserted. Preferably, a variant CDR1 domain comprises one or more,for example two, three, four or five substitutions, preferablyconservative substitutions. Examples of such CDR1 variant sequences arethe LV-CDR1 sequences identified in Tables 38, 39, 40, 41 and 42.Preferred CDR1 sequences include SEQ ID NOS: 33, 219, 226, 218, 326, 93,325, 391 and 394.

The sequences of preferred CDR2 domains are shown in SEQ ID NOS: 22, 25,28, 31, 34, 37, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85,88, 94, 112, 118 and 124. Other preferred CDR2 domains are variants ofthese sequences in which one or more amino acids within the sequencehave been deleted or, more preferably, substituted. Other preferred CDR2domains are variants of the sequences shown in one of SEQ ID NOS: 22,25, 28, 31, 34, 37, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82,85, 88, 94, 112, 118 and 124 in which one or more amino acid has beeninserted. Preferably, a variant CDR2 domain comprises one or more, forexample two, three, four or five substitutions, preferably conservativesubstitutions. Examples of such CDR2 variant sequences are the LV-CDR2sequences identified in Tables 38, 39, 40, 41 and 42. Preferred CDR2sequences include SEQ ID NOS: 382, 386, 333, 334, 34, 247 and 252.

The sequences of preferred CDR3 domains are shown in SEQ ID NOS: 23, 26,29, 32, 35, 38, 47, 50, 53, 56, 59, 62, 65, 65, 68, 71, 74, 77, 80, 83,86, 89, 95, 113, 119 and 125. Other preferred CDR3 domains are variantsof these sequence in which one or more amino acids within the sequencehave been deleted or, more preferably substituted. Other preferred CDR3domains are variants of the sequences shown in one of SEQ ID NOS: 23,26, 29, 32, 35, 38, 47, 50, 53, 56, 59, 62, 65, 65, 68, 71, 74, 77, 80,83, 86, 89, 95, 113, 119 and 125 in which one or more amino acid hasbeen inserted. Preferably, a variant CDR3 domain comprises one or more,for example two or three, conservative substitutions.

Conservative substitutions are shown in the following Table. Amino acidsin the same block in the second column and preferably in the same linein the third column may be substituted for each other.

ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

Examples of variant CDR3 sequences are the HV-CDR3 and LV-CDR3 sequencesidentified in Tables 38, 39, 40, 41 and 42. Preferred variant CDR3sequences are shown in SEQ ID NOS: 207, 209, 210, 35, 269, 252, 34, 322,323, 320, 341, 373 and 378.

Preferred antibodies comprise (i) the VH sequence shown in SEQ ID NO:39, or a variant thereof, and the VL sequence shown in SEQ ID NO: 42, ora variant thereof; (ii) the VH sequence shown in SEQ ID NO: 40, or avariant thereof, and the VL sequence shown in SEQ ID NO: 43, or avariant thereof; (iii) the VH sequence shown in SEQ ID NO: 41, or avariant thereof, and the VL sequence shown in SEQ ID NO: 44, or avariant thereof.

Other preferred antibodies comprise a heavy chain sequence selected fromthe sequences shown in SEQ ID NOS: 317, 318, 319, 369, 370, 371, 372 and397 and optionally a light chain sequence selected from SEQ ID NOS: 43,295, 294, 304, 347, 348, 357, 362, 406 and 418. More preferredantibodies include antibodies having the following combinations of heavyand light chain sequences: SEQ ID NOS: 319 and 43, SEQ ID NOS: 318 and295, SEQ ID NOS: 318 and 294, SEQ ID NOS: 317 and 304, SEQ ID NOS: 370and 347, SEQ ID NOS: 370 and 348, SEQ ID NOS: 371 and 348, SEQ ID NOS:372 and 348, SEQ ID NOS: 369 and 357, SEQ ID NOS: 370 and 362, SEQ IDNOS: 397 and 406. SEQ ID NOS: 397 and 418.

Variant antibodies may be obtained by any suitable method. Typicallyvariants with improved binding characteristics are selected by affinitymaturation.

In a preferred embodiment, the antibody is a recombinant or modifiedanti-ApoE-CTD antibody, e.g. a chimeric, humanised, deimmunised or an invitro generated antibody. The term “recombinant” or “modified” antibodyas used herein is intended to include all antibodies that are prepared,expressed, created or isolated by recombinant means, such as (i)antibodies expressed using a recombinant expression vector transfectedinto a host cell; (ii) antibodies isolated from a recombinant,combinatorial antibody library; (iii) antibodies isolated from an animal(e.g. a mouse) that is transgenic for human immunoglobulin genes; or(iv) antibodies prepared, expressed, created or isolated by any othermeans that involves splicing of human immunoglobulin gene sequences toother DNA sequences. Such recombinant antibodies include humanised, CDRgrafted, chimeric, deimmunised, in vitro generated antibodies and mayoptionally include constant regions derived from human germlineimmunoglobulin sequences.

An antibody according to the invention is a human antibody. The antibodymay be a chimeric antibody, a recombinant antibody, a humanisedantibody, a monoclonal antibody or a polyclonal antibody. Preferably theantibody is monoclonal.

The antibody may be conjugated to a functional moiety such as a drug,detectable moiety or a solid support.

Also within the scope of the invention are compositions comprising twoor more antibodies which bind different epitopes of ApoE-CTD. Theantibodies in the composition may bind overlapping epitopes. Antibodiesthat bind overlapping epitopes competitively inhibit the binding of eachother to ApoE-CTD.

The antibody is preferably monospecific, e.g. a monoclonal antibody, orantigen-binding fragment thereof. Bispecific and multivalent antibodiesare also provided, which bispecific or multivalent antibodies bind totwo or more different epitopes of ApoE-CTD.

An antibody of the invention may be joined to a binding moiety such asbiotin. For example, an antibody, preferably an IgG, may be biotinylatedby incubation with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. A biotinylated IgG preferably comprises from1 to 5 such as 2, 3 or 4 biotin groups.

An antibody of the invention may be in substantially isolated form. Theymay be mixed with carriers or diluents which will not interfere withtheir intended use and still be regarded as substantially isolated. Theymay also be in a substantially purified form, in which case they willgenerally comprise at least 90%, e.g. at least 95%, 98% or 99%, of thepolypeptides or dry mass of the preparation.

B. Methods For Identifying Antibodies

The invention also provides a method for identifying an antibodyaccording to the invention. The method typically comprises identifyingan antibody that binds to a polypeptide having the amino acid sequenceas shown in SEQ ID NO: 1 or the amino acid sequence of a part thereofand that binds to human plaques. Either or both binding assays may becarried out in the presence of VLDL. The methods generally compriseproviding a display library and screening the library to identify amember that encodes an antibody that binds to ApoE-CTD or a fragmentthereof and/or to human plaques, preferably in the presence of VLDL. Adisplay library is a collection of entities; each entity includes anaccessible antibody component and a recoverable component that encodesor identifies the antibody component. The antibody component can be ofany length, e.g., from three amino acids to over 300 amino acids forexample 30, 100 or 200 amino acids and is typically an antibodyfragment, preferably a Fab fragment. In a selection, the antibodycomponent of each member of the library is probed with ApoE-CTD and ifthe antibody component binds to ApoE-CTD or fragment thereof, thedisplay library member is identified, typically by retention on asupport. Display library members that bind ApoE-CTD may also typicallytested for binding to ApoE-NTD (negative selection).

Retained display library members are recovered from the support andanalysed. The analysis can include amplification and a subsequentselection under similar or dissimilar conditions. For example, positiveand negative selections can be alternated. The analysis can also includedetermining the amino acid sequence of the antibody component andpurification of the antibody component for detailed characterisation.

A variety of formats can be used for display libraries and any suitableformat may be used in a method of the invention. Preferred formats arephage display and cell-based display such as yeast display.

In phage display, the candidate antibodies are typically covalentlylinked to bacteriophage coat protein. The linkage results fromtranslation of a nucleic acid encoding the candidate antibodies fused tothe coat protein. The linkage can include a flexible peptide linker, aprotease site, or an amino acid incorporated as a result of suppressionof a stop codon. Phage display is described, for example, in Ladner etal., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/02809; WO 90/09690; de Haard et al. (1999) J. Biol.Chem. 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4: 1-20;Hoogenboom et al. (2000) Immunol. Today 2:371-8; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991)Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol.267:129-49; Hoogenboom et al. (1991) Nuc. Acids Res. 19:4133-4137;Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and Lee etal. (2003) Trends In Biotechnology 21: 45-52.

Phage display systems have been developed for filamentous phage (phagefl, fd and M13) as well as other bacteriophage (e.g., T7 Bacteriophageand lambdoid phages; see, e.g., Santini (1998) J. Mol. Biol.282:125-135; Rosenberg et al. (1996) Innovations 6:1-6; Houshmet et al.(1999) Anal. Biochemn. 268:363-370). The filamentous phage displaysystems typically use fusions to a minor coat protein, such as gene IIIprotein, and gene VIII protein, a major coat protein, but fusions toother coat proteins such as gene VI protein, gene VII protein, gene IXprotein, or domains thereof can also been used (see, e.g., WO 00/71694).In a preferred embodiment, the fusion is to a domain of the gene IIIprotein, e.g., the anchor domain or “stump,” (see, e.g., U.S. Pat. No.5,658,727 for a description of the gene III protein anchor domain).

The valency of the candidate polypeptides can also be controlled.Cloning of the sequence encoding the polypeptide component into thecomplete phage genome results in multivariant display since allreplicates of the gene III protein are fused to the polypeptidecomponent. For reduced valency, a phagemid system can be utilised. Inthis system, the nucleic acid encoding the polypeptide component fusedto gene III is provided on a plasmid, typically of length less than 700nucleotides. The plasmid includes a phage origin of replication so thatthe plasmid is incorporated into bacteriophage particles when bacterialcells bearing the plasmid are infected with helper phage, e.g., M13K07.The helper phage provides an intact copy of gene III and other phagegenes required for phage replication and assembly. The helper phage hasa defective origin such that the helper phage genome is not efficientlyincorporated into phage particles relative to the plasmid that has awild type origin.

Bacteriophage displaying the candidate antibodies can be grown andharvested using standard phage preparatory methods, e.g., PEGprecipitation from growth media.

After selection of individual display phages, the nucleic acid encodingthe selected candidate antibodies can be obtained by infecting cellsusing the selected phages. Individual colonies or plaques can be picked,the nucleic acid isolated and sequenced.

In a screening procedure to obtain ApoE-CTD binders according to thisinvention, a display library is contacted with and allowed to bind atarget ApoE-CTD molecule, usually immobilised on a solid support.Non-binders are separated from binders. In various ways, the bound phageare liberated from the ApoE-CTD, collected and amplified. Since thephage can be amplified through infection of bacterial cells, even a fewbinding phage are sufficient to reveal the gene sequence that encodes abinding entity. Using these techniques it is possible to recover abinding phage that is about 1 in 20 million in the population. One ormore libraries, displaying 10-20 million or more potential bindingpolypeptides each, can be rapidly screened to find high-affinityApoE-CTD binders. When the selection process works, the diversity of thepopulation falls with each round until only good binders remain, i.e.,the process converges. Typically, a phage display library will containseveral closely related binders (10 to 50 binders out of 10 million).Indications of convergence include increased binding (measured by phagetiters) and recovery of closely related sequences.

In a cell-display library the candidate antibodies are displayed on thesurface of a cell, e.g., a eukaryotic or prokaryotic cell. Exemplaryprokaryotic cells include E. coli cells, B. subtilis cells and spores(see, e.g., Lu et al. (1995) Biotechnology 13:366). Exemplary eukaryoticcells include yeast (e.g., Saccharomyces cerevisiae, Schizosaccharmycespombe, Hanseulla, or Pichia pastoris). Yeast surface display isdescribed in, for example, Boder and Wittrup (1997) Nature Biotech.15:553-557. A yeast display system that may be used to displayimmunoglobulin proteins such as Fab fragments, and yeast mating may beused to generate combinations of heavy and light chains.

Yeast display has clear advantages over phage display in the applicationof affinity maturation of anti-ApoE-CTD antibodies. The most importantadvantage is that FACS selection may be used to quantitatively sort eachyeast cell for its antigen binding. It is also possible to performnormalized selection so that variations in display level can becorrected, thus avoiding selection on the basis of avidity. This isparticularly important when using a multivalent target antigen.

The display library may be a ribosome display library. In a ribosomedisplay library mRNA and the candidate antibody encoded by the RNA canbe physically associated by stabilising ribosomes that are translatingthe mRNA and have the nascent polypeptide still attached. Typically,high divalent Mg²⁺ concentrations and low temperature are used. See,e.g., Mattheakis at al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 andHanes at al. (2000) Nature Biotech. 18:1287-92; Hans at al. (2000)Methods Enzymol. 328-404-30 and Schaffitzel et al. (1999) J. Immunol.Methods 231:119-35.

Another display library format utilises peptide-nucleic acid fusions.Polypeptide-nucleic acid fusions can be generated by the in vitrotranslation of mRNA that include a covalently attached puromycin group,e.g., as described in Roberts and Szostak (1997) Proc. Acad. Sci. USA94:12297-12302, and U.S. Pat. No. 6,207,446. The mRNA can then bereverse transcribed into DNA and crosslinked to the polypeptide.

Another display format that may be used is a non-biological display inwhich the antibody component is attached to a non-nucleic acid tag thatidentifies the antibody. For example, the tag can be a chemical tagattached to a bead that displays the antibody or a radiofrequency tag(See, e.g., U.S. Pat. No. 5,874,214).

A parental binding domain is selected to serve as a structural templatefor the candidate antibodies. The binding domain may be a naturallyoccurring or synthetic protein, or a region or domain of a protein suchas an immunoglobulin. The parental binding domain may be selected basedon knowledge of a known interaction between the parental binding domainand ApoE-CTD but, but this is not critical. In fact, it is not essentialthat the parental binding domain have any affinity for ApoE-CTD at all:its purpose is to provide a structure from which a library can begenerated, which library will include one or more candidate antibodiesthat bind specifically to ApoE-CTD.

The candidate antibodies may be Fab fragments, single chain Fv molecules(scFV), single domain antibodies, camelid antibodies and camelizedantibodies.

In a preferred embodiment, the parental binding domain comprises animmunoglobulin domain with antigen-binding activity, such as scFv, Fabor IgG. A typical display library displays candidate polypeptides thatinclude a VH domain and a VL domain. As in the case of the Fab and otherformats, the displayed antibody can include a constant region as part ofa light or heavy chain. In one embodiment, each chain includes oneconstant region, e.g. as in the case of a Fab. In other embodiments,additional constant regions are displayed.

Display libraries are particularly useful, for example for identifyinghuman or “humanised” antibodies that recognise human antigens. The invitro display selection process surmounts the inability of a normalhuman immune system to generate antibodies against self-antigens.

Antibody libraries can be constructed by a number of processes (see,e.g. de Haard et al (1999) J. Biol. Chem. 274:18218-30; Hoogenboom et al(1998) Immunotechnology 4:1-20, and Hoogenboom et al (2000) Immunol.Today 21:371-8). Further, elements of each process can be combined withthose of other processes. The processes can be used such that variationis introduced into a single immunoglobulin domain (e.g. VH or VL) orinto multiple immunoglobulin domains (e.g. VH and VL). The variation canbe introduced into an immunoglobulin variable domain, e.g. in the regionof one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4, referring tosuch regions of either and/or both of heavy and light chain variabledomains. In one embodiment, variation is introduced into all three CDRsof a given variable domain. In another preferred embodiment, thevariation is introduced into CDR1 and CDR2, e.g. of a heavy chainvariable domain. Any combination is feasible.

In a preferred embodiment the parental domain comprises the CDR3sequence shown in any one of SEQ ID NOS: 23, 26, 29, 32, 35, 38, 47, 50,53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 95, 113, 119 and125. Amino acid substitutions at one or both of positions 2 and 3 of SEQID NO: 23 or 26 are preferred variations in candidate antibodies.Examples of variant VH-CDR3 sequences generated by antibody spiking areidentified in Tables 38, 39, 40, 41 and 42. Preferred CDR3 sequences areshown in SEQ ID NOS: 207, 209, 210, 320, 322, 323 and 373. Consensussequences for preferred CDR3 sequences are shown in SEQ ID NOS: 512,513, 514, 515, 516, 517 and 20.

Examples of variant VL-CDR3 sequences generated by light chain shufflingare also shown in Tables 38, 39, 40, 41 and 42. Preferred VL-CDR3sequences are shown in SEQ ID NOS: 35, 269, 275, 268, 341 and 378.

The parental domain preferably also comprises the other components ofthe VH chain, and optionally a VL chain, or the other components of theVL chain, and optionally a VH chain.

A second preferred parental domain comprises a CDR1 and/or CDR2 domainwith the sequence shown in any one of SEQ ID NOS: 21, 24, 27, 30, 33,36, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 93, 111,117 and 123 or in SEQ ID NOS: 22, 25, 28, 31, 34, 37, 46, 49, 52, 55,58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 94, 112, 118 and 124. Thecandidate polypeptides may be generated by DNA shuffling the CDR1 and/orCDR2 domains. Examples of variant VL-CDR1 and VL-CDR2 sequences areidentified in Tables 38, 39, 40, 41 and 42. Preferred CDR1 sequences areshown in SEQ ID NOS: 33, 219, 226, 218, 93, 325, 326, 391 and 394.Preferred CDR2 sequences are shown in SEQ ID NOS: 34, 247, 252, 333,334, 382 and 386.

A third preferred parental domain comprises the VL sequence shown in anyone of SEQ ID NOS: 42, 43, 44, 151, 157, 159 and 161. Candidatepolypeptides are typically generated by DNA shuffling of the entire VLsequence. Examples of shuffled light chain sequences are identified inTables 38, 39, 40, 41 and 42. Preferred shuffled light chain sequencesare shown in SEQ ID NOS: 43, 295, 294, 304, 347, 348, 357, 362, 406 and418.

In one process, antibody libraries are constructed by inserting diverseoligonucleotides that encode CDRs into the corresponding regions of thenucleic acid. The oligonucleotides can be synthesized using monomericnucleotides or trinucleotides. For example, Knappik et al ((2000) J Mol.Biol. 296:57-86) describes a method for constructing CDR encodingoligonucleotides using trinucleotide synthesis and a template withengineered restriction sites for accepting the oligonucleotides.

In another process, an animal, e.g. a rodent, is immunised with theApoE-CTD. The animal is optionally boosted with the antigen to furtherstimulate the response. Then spleen cells are isolated from the animal,and nucleic acid encoding VH and/or VL domains is amplified and clonedfor expression in the display library.

In yet another process, antibody libraries are constructed from nucleicacid amplified from naïve germline immunoglobulin genes. The amplifiednucleic acid includes nucleic acid encoding the VH and/or VL domain.Sources of immunoglobulin-encoding nucleic acids are described below.Amplification can include PCR, e.g. with primers that anneal to theconserved constant region, or another amplification method.

Nucleic acid encoding immunoglobulin domains can be obtained from theimmune cells of, e.g. a human, a primate, mouse, rabbit, camel orrodent. In one example, the cells are selected for a particularproperty. B cells at various stages of maturity can be selected. Inanother example, the B cells are naïve.

In one embodiment, fluorescent-activated cell sorting (FACS) is used tosort B cells that express surface-bound IgM, IgD or IgG molecules.Further, B cells expressing different isotypes of IgG can be isolated.In another preferred embodiment, the B or T cell is cultured in vitro.The cells can be stimulated in vitro, e.g. by culturing with feedercells or by adding mitogens or other modulatory reagents, such asantibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate,bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin orpokeweed mitogen.

In still another embodiment, the cells are isolated from a subject thathas an immunological disorder, e.g. systemic lupus erythematosus (SLE),rheumatoid arthritis, vasculitis, Sjogren syndrome, systemic sclerosisor anti-phospholipid syndrome. The subject can be a human or an animal,e.g. an animal model for the human disease, or an animal having ananalogous disorder. In yet another embodiment, the cells are isolatedfrom a transgenic non-human animal that includes a human immunoglobulinlocus.

In one preferred embodiment, the cells have activated a program ofsomatic hypermutation. Cells can be stimulated to undergo somaticmutagenesis of immunoglobulin genes, for example, by treatment withanti-immunoglobulin, anti-CD40 and anti-CD38 antibodies (see, e.g.Bergthorsdottir et al (2001) J. Immunol 166:2228). In anotherembodiment, the cells are naïve.

The nucleic acid encoding an immunoglobulin variable domain can beisolated from a natural repertoire by the following exemplary method.First, RNA is isolated from the immune cell. Full length (i.e. capped)mRNAs are separated (e.g. by degrading uncapped RNAs with calfintestinal phosphatase). The cap is then removed with tobacco acidpyrophosphatase and reverse transcription is used to produce the cDNAs.

The reverse transcription of the first (antisense) strand can be done inany manner with any suitable primer. See, e.g. de Haard et al (1999) J.Biol. Chem. 274:18218-30. The primer binding region can be constantamong different immunoglobulins, e.g. in order to reverse transcribedifferent isotypes of immunoglobulin. The primer binding region can alsobe specific to a particular isotype of immunoglobulin. Typically, theprimer is specific for a region that is 3′ to a sequence encoding atleast one CDR. In another embodiment, poly-dT primers may be used (andmay be preferred for the heavy-chain genes).

A synthetic sequence can be ligated to the 3′ end of the reversetranscribed strand. The synthetic sequence can be used as a primerbinding site for binding of the forward primer during PCR amplificationafter reverse transcription. The use of the synthetic sequence canobviate the need to use a pool of different forward primers to fullycapture the available diversity.

The variable domain-encoding gene is then amplified, e.g. using one ormore rounds. If multiple rounds are used, nested primers can be used forincreased fidelity. The amplified nucleic acid is then cloned into adisplay library vector.

Any method for amplifying nucleic acid sequences may be used foramplification. Methods that maximise, and do not bias, diversity arepreferred. Suitable techniques for nucleic acid amplification includethe polymerase chain reaction (PCR), transcription-based methods thatutilise RNA synthesis by RNA polymerases to amplify nucleic acid (Sarkeret al (1989) Science 244:331-34), NASBA (U.S. Pat. Nos. 5,130,238;5,409,818; and 5,554,517) which utilises cycles of transcription,reverse-transcription and RnaseH-based degradation to amplify a DNAsample, rolling circle amplification (RCA; U.S. Pat. No. 6,143,495) andstrand displacement amplification (SDA; U.S. Pat. No. 5,624,825).

After a first set of binding antibodies is identified, the sequenceinformation can be used to design other libraries biased for membershaving additional desired properties, e.g., discrimination betweenApoE-CTD and full-length ApoE, preferably VLDL-associated ApoE. Suchtechniques make it possible not only to screen a large number ofpotential binding antibodies but also make it practical to repeat thebinding/elution cycles and to build secondary, biased libraries forscreening analog-displaying packages that meet initial criteria Usingthese techniques, a biased library may be screened to reveal membersthat bind tightly (i.e., with high affinity) under the screeningconditions.

Thus, in one preferred embodiment, display library technology may beused in an iterative mode. A first display library is used to identifyone or more antibodies that bind ApoE-CTD and/or human plaques. Theseidentified antibodies are then varied using a mutagenesis method to forma second display library. Higher affinity polypeptides are then selectedfrom the second library, e.g., by using higher stringency or morecompetitive binding and washing conditions.

In affinity maturation protocols, the variation is preferably generatedby amino acid substitutions but may also result from deletion oraddition of amino acids.

The amino acid substitutions may be those which are expected to alterthe binding properties of the domain without significantly altering itsstructure, at least for most substitutions. It is preferred that theamino acid positions that are selected for variation (variable aminoacid positions) will be surface amino acid positions, that is, positionsin the amino acid sequence of the domains which, when the domain is inits most stable conformation, appear on the outer surface of the domain(i.e., the surface exposed to solution). Most preferably the amino acidpositions to be varied will be adjacent or close together, so as tomaximise the effect of substitutions. In addition, extra amino acids canbe added into the structure of the parental binding domain.

In some implementations, the mutagenesis is targeted to regions known orlikely to be at the binding interface. Mutagenesis can be directed tothe CDR regions of the heavy or light chains as described herein.Further, mutagenesis can be directed to framework regions near oradjacent to the CDRs. Mutagenesis can also be limited to one or a few ofthe CDRs, e.g., to make precise step-wise improvements.

Effective affinity maturation requires 4 components (i) therediversification of lead antibody genes (ii) display on either phage oryeast (iii) affinity selection (iv) screening of clones for improvedaffinity.

Alignment of Fabs showing the required binding properties, for exampleusing a BLAST algorithm (e.g. Karlin and Altschul (1993) PNAS USA 90:5873-5787) may be used to identify conserved residues in the CDRdomains. Sequence similarity amongst the CDR loops may allow aprediction of the direct involvement of any amino acid in antibodyaffinity or specificity.

For example, the VH-CDR3 loops of antibodies 807A-M0028-B02 and807A-M0027-E11 (SEQ ID NOS: 23 and 26) show striking similarity and showconsensus over 4/6 amino acids (SEQ ID NO: 20). This suggests that theVH-CDR3 plays a role in antibody affinity and specificity.

An optimal antibody mutagenesis strategy introduces a minimal number ofmutations at functionally relevant positions. This is achieved by bothtargeted and non-targeted mutagenesis procedures. Non-targetedmutagenesis procedures include chain shuffling which introduces largeblock changes in antibodies by rediversifying the whole VL gene or theVH CDR1-2 fragment. Typically, the VH-CDR3 loop is left untouched, as itmay make significant contributions to binding affinity and specificity.Examples of chain shuffling are described in the following documents:Marks et al., (1992) Nature Biotech 10: 779-783, Schier et al., (1996)J. Mol. Biol. 255, 28-43, Park et al., (2000) BBRC. 275. 553-557 andChames et al., (2002) J. Immunol. 1110-1118. Although chain shuffling isa well-validated technique (particularly for antibodies with a lowstarting affinity) a possible disadvantage is that by making such largeblock changes in the antibody molecule that there may be an increasedchance of disrupting multiple favourable contacts. However this could becompensated by the loss of unfavourable contacts or the generation ofnew contacts.

Other exemplary non-targeted mutagenesis techniques include: error-pronePCR (Leung et al. (1989) Technique 1:11-15), recombination, DNAshuffling using random cleavage (Stemmer (1994) Nature 389-391; termed“nucleic acid shuffling”), RACHITT™ (Coco at al. (2001) Nature Biotech.19:354), site-directed mutagenesis (Zooler et al. (1987) Nucl. AcidsRes. 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) MethodsEnzymol. 208:564-586) and incorporation of degenerate oligonucleotides(Griffiths at al. (1994) EMBO J. 13:3245).

Targeted mutagenesis procedures include hot spot mutagenesis,parsimonious mutagenesis, saturation mutagenesis, domain randomisationand domain walking. CDR mutagenesis can be done in a stepwise mannerthat is target CDR1, select an optimised loop and subsequently targetCDR2 etc. The single most naturally diverse loop is the VH-CDR3 and itis generally accepted that as this loop is situated centrally in theantibody combining site that this is a critical determinant of antibodyspecificity and affinity. This means that there is a strong case forspecifically targeting this loop.

In one example of iterative selection, the methods described herein areused to first identify an antibody from a display library that bindsApoE-CTD with at least a minimal binding specificity for a target or aminimal activity, e.g., an equilibrium dissociation constant for bindingof greater than 1 nM, 10 nM, or 100 nM, and which binds human plaquesand or/which retains binding activity in the presence of VLDL-associatedApoE. The nucleic acid sequence encoding the initial identified antibodyis used as a template nucleic acid for the introduction of variations,e.g., to identify a second polypeptide that has enhanced properties(e.g., binding affinity, kinetics, or stability) relative to the initialantibody.

Antibodies according to the present invention may be isolated usingdisplay technology, in a manner to identify ApoE-CTD binding antibodiesexhibiting particular preselected properties of binding and release.According to this methodology, two solution conditions may bepreselected, i.e., binding conditions and release conditions. Thebinding conditions are a set of solution conditions under which isdesired that a discovered antibody will bind the target ApoE-CTD; therelease conditions are a set of solution conditions under which it isdesired that a discovered antibody will not bind the ApoE-CTD (i.e. willdissociate from ApoE-CTD). The two conditions may be selected to satisfyany criterion of the practitioner, such as ease of attaining theconditions, compatibility with other purification steps, lowered cost ofswitching between conditions compared to other affinity media, etc.Preferably, the two solution conditions are selected so as not toadversely affect the stability or activity of the target proteinApoE-CTD and so as to differ significantly with respect to at least onesolution parameter. For example, in conducting the screening forsuitable binding peptides described herein, binders are selected thatdissociated from the target in the presence of an ethyleneglycol-containing buffer, or conditions of lowered pH (i.e. pH 2), orcombinations of those conditions, which differ from the conditionsemployed for binding. Another parameter that can be advantageouslyvaried is the concentration of a salt, for example NaCl, in the bindingand elution buffers.

An antibody which binds to ApoE-CTD typically has a minimal bindingspecificity for ApoE-CTD, for example an equilibrium constant forbinding of greater than 1 nM, 10 nM or 100 nM.

Since a slow dissociation rate can be predictive of high affinity,particularly with respect to interactions between antibodies and theirtargets, methods of off-rate selection can be used to isolate antibodieswith a desired kinetic dissociation rate (i.e., reduced) for a bindinginteraction to an ApoE-CTD.

To select for slow dissociating antibodies from a display library, thelibrary is contacted to an immobilised target, preferably ApoE-CTD. Theimmobilised target is then washed with a first solution that removesnon-specifically or weakly bound antibodies. Then the immobilised targetis eluted with a second solution that includes a saturation amount offree target, i.e., replicates of the target that are not attached to theparticle. The free target binds to antibodies that dissociate from thetarget. Rebinding is effectively prevented by the saturating amount offree target relative to the much lower concentration of immobilisedtarget.

The second solution can have solution conditions that are substantiallyphysiological or that are stringent. Typically, the solution conditionsof the second solution are identical to the solution conditions of thefirst solution. Fractions of the second solution are collected intemporal order to distinguish early from late fractions. Later fractionsinclude biomolecules that dissociate at a slower rate from the targetthan biomolecules in the early fractions.

Further, it is also possible to recover display library members thatremain bound to the target even after extended incubation. These caneither be dissociated using chaotropic conditions or can be amplifiedwhile attached to the target. For example, phage bound to the target canbe contacted to bacterial cells.

The ApoE-CTD used in a method of the invention may be in any suitableform. ApoE-CTD typically has the amino acid sequence set out in SEQ IDNO. 1 or the amino acid sequence of a fragment thereof. The fragment ofApoE-CTD is typically at least three amino acids in length, preferablyat least five, six, seven or eight amino acids in length and morepreferably at least 10, 12 or 16 amino acids in length. Examples ofsuitable fragments are set out in SEQ ID NOS: 2 to 19. Preferredfragments are those having a sequence shown in any one of SEQ ID NOs: 2,5, 7, 9, 10, 12, 13, 14, 15, 16 and 17. One or more ApoE-CTD peptide maybe used in a screening assay of the invention.

The ApoE-CTD polypeptides are generally produced by recombinant means.Urea-denatured ApoE-CTD which has been recombinantly or naturallyproduced may be used in a method of the invention. Candidatepolypeptides may additionally or alternatively be screened for bindingto CTD in a polymeric form (ApoE-CTD binds to fibrils). Binding to acomplex of ApoE-CTD and AD may also be monitored.

ApoE-CTD may be cleaved from recombinant or naturally occurring ApoE,for example by the action of thrombin.

The ApoE-CTD polypeptide or peptide may be immobilised on a support.Typically immobilisation is achieved by tagging or biotinylating thepolypeptide for capture onto a surface. For example, the ApoE-CTD maycomprise an S—S biotin group for attachment to streptavadin (for exampleon streptavadin-coated magnetic beads). Alternatively the ApoE-CTD maycomprise a cysteine residue for coupling to a BSA carrier forimmobilisation (for example on plastic). In this way a “CTD-coated chip”may be produced. Binding of candidate polypeptide to a CTD-coated chipmay be analysed by BIACORE analysis.

Display library members may also be screened for binding to humanplaques.

The display library screening methods described herein preferablyinclude a selection or screening process that discards display librarymembers that bind to a non-target molecule. Examples of non-targetmolecules include: streptavidin and (ii) ApoE-NTD.

In one implementation, a so-called “negative selection” step is used todiscriminate between the target and related non-target molecule and arelated, but distinct non-target molecule. The display library or a poolthereof is contacted to the non-target molecule. Members of the samplethat do not bind the non-target are collected and used in subsequentselections for binding to the target molecule or even for subsequentnegative selections. The negative selection step can be prior to orafter selecting library members that bind to the target molecule.

In another implementation, a screening step is used. After displaylibrary members are isolated for binding to the target molecule, eachisolated library member is tested for its ability to bind to anon-target molecule (e.g., a non-target listed above). For example, ahigh-throughput ELISA screen can be used to obtain this data. The ELISAscreen can also be used to obtain quantitative data for binding of eachlibrary member to the target. The non-target and target binding data arecompared (e.g. using a computer and software) to identify librarymembers that specifically bind to the target MHC-peptide complex.

An antibody or antibody fragment of the invention may bind to ApoE inthe presence of VLDL or other lipoprotein particles. An antibody of theinvention typically binds to ApoE with a minimal binding specificity forApoE-CTD, e.g. an equilibrium constant for binding of greater than 1 nMor 100 nM in the presence of VLDL. The VLDL may be present in anysuitable form. For example human plasma may be added to the bindingassay. Up to 50% human plasma may be added to the assay, for example upto 10%, up to 20%, up to 30% or up to 40% human plasma may be included.

The candidate polypeptides may also be screened for binding to ApoE-NTD.ApoE-NTD may be produced recombinantly or may be cleaved fromrecombinant or naturally occurring ApoE, for example by the action ofthrombin.

In one embodiment, the candidate polypeptides may be screened forbinding to astrocytes. It is preferred but not essential, that theselected polypeptides do not bind to astrocytes or bind with a muchlower affinity to astrocytes than to ApoE-CTD, for example a two-fold,five-fold, 10-fold, 20-fold or 50-fold lower affinity.

After selecting candidate display library members that bind to ApoE-CTD,each candidate display library member may be further analysed, e.g. tofurther characterise its binding properties for the target. Eachcandidate display library member can be subjected to one or moresecondary screening assays. The assay can be for a binding property, acatalytic property, a physiological property (e.g. cytotoxicity, renalclearance, immunogenicity), a structural property (e.g. stability,conformation, oligomerisation state) or another functional property. Thesame assay can be used repeatedly, but with varying conditions, e.g. todetermine pH, ionic or thermal sensitivities.

As appropriate, the assays can use the display library member directly,a recombinant antibody produced from the nucleic acid encoding adisplayed antibody, or a synthetic antibody synthesised based on thesequence of a displayed antibody. The assays preferably comprisedetermining whether or not an antibody that binds ApoE-CTD also binds tohuman plaques, or whether it binds to ApoE-CTD in the presence of VLDL.Exemplary assays for binding properties include ELISA, homogeneousbinding assays such as fluorescence resonance energy transfer (FRET) andalpha-screen, surface plasmon resonance (SPR), protein assays andcellular assays.

Antibodies encoded by a display library can also be screened for abinding property using an ELISA. For example, each candidate antibodythat binds ApoE-CTD is brought into contact with a microtitre platewhose bottom surface has been coated with ApoE-CTD, VLDL or ApoE-NTD.The plate is washed with buffer to remove non-specifically boundpolypeptides. Then the amount of the polypeptide bound to the plate isdetermined by probing the plate with an antibody that can recognise thepolypeptide, e.g. a tag or constant portion of the polypeptide. Theantibody is linked to an enzyme such as alkaline phosphatase, whichproduces a colorimetric product when appropriate substrates areprovided. The polypeptide can be purified from cells or assayed in adisplay library format, e.g. as a fusion to a filamentous bacteriophagecoat. In another version of the ELISA, each polypeptide of a diversitystrand library is used to coat a different well of a microtitre plate.The ELISA then proceeds using a constant target molecule to query eachwell. A polypeptide specifically binds ApoE-CTD in an ELISA if itdisplays at least 2× background on ApoE-CTD but less than 1× backgroundon negative control proteins such as ApoE-NTD or streptavidin.

A homogeneous binding assay is an assay in which the binding interactionof candidate antibody with a target can be analysed after all componentsof the assay are added without additional fluid manipulations beingrequired. For example, fluorescence resonance energy transfer (FRET) canbe used as a homogenous assay (see, for example, Lakowicz et al, U.S.Pat. No. 5,631,169). Another example of a homogenous assay is AlphaScreen (Packard Bioscience, Meriden, Conn., USA).

The homogenous assays can be performed while the candidate polypeptideis attached to the display library vehicle, e.g. a bacteriophage.

The binding interaction of a molecule isolated from a display libraryand a target can be analysed using Surface Plasmon Resonance (SPR). SPRor Biomolecular Interaction Analysis (BIA) detects biospecificinteractions in real time, without labelling any of the interactants.Changes in the mass at the binding surface (indicative of a bindingevent) of the BIA chip result in alterations of the refractive index oflight near the surface (the optical phenomenon of surface plasmonresonance (SPR)). The changes in the refractivity generate a detectablesignal, which are measured as an indication of real-time reactionsbetween biological molecules. Methods for using SPR are described, forexample, in Szabo et al (1995) Curr. Opin. Struct. Biol. 5:699-705 andon-line resources provided by BIAcore International AB (Uppsala,Sweden).

Information from SPR can be used to provide an accurate and quantitativemeasure of the equilibrium dissociation constant (K_(D)), and kineticparameters, including K_(on) and K_(off), for the binding of abiomolecule to a target. Such data can be used to compare differentbiomolecules. For example, proteins encoded by nucleic acid selectedfrom a library of diversity strands can be compared to identifyindividuals that have high affinity for the target or that have a slowK_(off). This information can also be used to develop structure-activityrelationships (SAR). For example, the kinetic and equilibrium bindingparameters of matured versions of a parent protein can be compared tothe parameters of the parent protein. Variant amino acids at givenpositions can be identified that correlate with particular bindingparameters, e.g. high affinity and slow K_(off). This information can becombined with structural modelling (e.g. using homology modelling,energy minimisation or structure determination by crystallography orNMR). As a result, an understanding of the physical interaction betweenthe protein and its target can be formulated and used to guide otherdesign processes.

Antibodies identified from the display library can be immobilised on asolid support, for example, on a bead or an array. For a protein array,each of the polypeptides is immobilised at a unique address on asupport. Typically, the address is a two-dimensional address. Proteinarrays are described below (see, e.g. “Diagnostics”).

A candidate antibody identified as binding to ApoE-CTD can be screenedby transforming vector nucleic acid sequences that encode the antibodyinto a host cell such that antibodies are produced within the cell,secreted from the cell, or attached to the cell surface. The cells canbe screened for antibodies that bind to ApoE-CTD, for example bydetecting a change in a cellular phenotype or a cell-mediated activity.For example, the activity may be cell or complement-mediatedcytotoxicity.

In another embodiment, the library of cells is in the form of a cellulararray. The cellular array can likewise be screened for any appropriatedetectable activity.

C. Producing an Antibody

Standard recombinant nucleic acid methods can be used to express anantibody of the invention. Generally, a nucleic acid sequence encodingthe antibody is cloned into a nucleic acid expression vector. Of course,if the antibody includes multiple polypeptide chains, each chain must becloned into an expression vector, e.g. the same or different vectors,that are expressed in the same or different cells. If the antibodyfragment is sufficiently small, i.e. has less than 50 amino acids, itcan be synthesised using automated organic synthetic methods. Methodsfor producing antibodies are also provided below.

The expression vector for expressing the antibody ligand can include, inaddition to the segment encoding the polypeptide ligand or fragmentthereof, regulatory sequences, including for example, a promoter,operably linked to the nucleic acid(s) of interest. Large numbers ofsuitable vectors and promoters are known to those of skill in the artand are commercially available for generating the recombinant constructsof the present invention. The following vectors are provided by way ofexample. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG and pSVL (Pharmacia). One preferred class of preferredlibraries is the display library, which is described below.

Methods well known to those skilled in the art can be used to constructvectors containing an antibody of the invention and appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Sambrook & Russell, Molecular Cloning: A Laboratory Manual,3^(rd) Edition, Cold Spring Harbor Laboratory, NY (2001) and Ausubel etal, Current Protocols in Molecular Biology (Greene Publishing Associatesand Wiley Interscience, NY (1989)). Promoter regions can be selectedfrom any desired gene using CAT (chloramphenicol transferase) vectors orother vectors with selectable markers. Two appropriate vectors arepKK232-8 and pCM7. Particular named bacterial promoters include lacIlacZ, T3, T7, gpt, lambda P and trc. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, mouse metallothionein-I and various art-known tissuespecific promoters.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g. the ampicillin resistance gene of E. coli and S. cerevisiaeauxotrophic markers (such as URA3, LEI2, HIS3 and TRPl genes), and apromoter derived from a highly expressed gene to direct transcription ofa downstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase or heat shock proteins, among others.The polynucleotide of the invention is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, anucleic acid of the invention can encode a fusion protein including anN-terminal identification peptide imparting desired characteristics,e.g. stabilisation or simplified purification of expressed recombinantproduct. Useful expression-vectors for bacteria are constructed byinserting a polynucleotide of the invention together with suitabletranslation initiation and termination signals, optionally in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces and Staphylococcus, although others may also be employed asa matter of choice.

As a representative but non-limiting example, useful expression vectorsfor bacteria can comprise a selectable marker and bacterial origin ofreplication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega, Madison, Wis., USA).

The present invention further provides host cells containing the vectorsof the present invention, wherein the nucleic acid has been introducedinto the host cell using known transformation, transfection or infectionmethods. For example, the host cells can include members of a libraryconstructed from the diversity strand. The host cell can be a eukaryotichost cell, such as a mammalian cell, a lower eukaryotic host cell, suchas a yeast cell, or the host cell can be a prokaryotic cell, such as abacterial cell. Introduction of the recombinant construct into the hostcell can be effected, for example by calcium phosphate transfection,DEAE, dextran mediated transfection or electroporation (Davis, L. et al,Basic Methods in Molecular Biology (1986)).

Any host/vector system can be used to identify one or more of the targetelements of the present invention. These include, but are not limitedto, eukaryotic hosts such as HeLa cells, CV-1 cells, COS cells, Sf9cells and HEK293T cells as well as prokaryotic hosts such as E. coli andB. subtilis. The most preferred cells are those which do not normallyexpress the particular reporter polypeptide or protein or whichexpresses the reporter polypeptide or protein at low natural level.

The host of the present invention may also be a yeast or other fungi. Inyeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol, 2, Ed. Ausubel et al, Greene Publish. Assoc. & WileyInterscience, Ch. 13 (1988); Grant et al (1987) “expression andSecretion Vectors for Yeast”, Methods Enzymol. 153:516-544 (1987); andThe Molecular Biology of the Yeast Saccharomyces, Eds. Strathem et al,Cold Spring Harbor Press, Vols. I and II (1982).

The host of the invention may also be a prokaryotic cell such as E.coli, other enterobacteriaceae such as Serratia marescans, bacilli,various pseudomonads or other prokaryotes which can be transformed,transfected and/or infected.

The present invention further provides host cells genetically engineeredto contain the antibodies of the invention. For example, such host cellsmay contain nucleic acids of the invention introduced into the host cellusing known transformation, transfection or infection methods. Thepresent invention still further provides host cells geneticallyengineered to express the polynucleotides of the invention, wherein suchpolynucleotides are in operative association with a regulatory sequenceheterologous to the host cell which drives expression of the antibodiesin the cell.

The host cell can be a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell.

Introduction of the recombinant construct into the host cell can beeffected by calcium phosphate transfection, DEAB, dextran mediatedtransfection or electroporation (David, L. et al, (1986) Basic Methodsin Molecular Biology). The host cells containing one of thepolynucleotides of the invention can be used in a conventional manner toproduce the gene product encoded by the isolated fragment (in the caseof an ORF).

Any on suitable host/vector system can be used to express one or more ofthe diversity antibodies of the present invention. Various mammaliancell culture systems can also be employed to express recombinantantibodies.

Antibodies, e.g. Fabs, can be produced in bacterial cells, e.g. E. colicells. For example, if the Fab is encoded by sequences in a phagedisplay vector that includes a suppressible stop codon between thedisplay entity and a bacteriophage protein (or fragment thereof), thevector nucleic acid can be shuffled into a bacterial cell that cannotsuppress a stop codon. In this case, the Fab is not fused to the geneImI protein and is secreted into the media Antibodies can also beproduced in eukaryotic cells. In one embodiment, the antibodies (e.g.scFvs) are expressed in a yeast cell such as Pichia (see, e.g. Powers etal (2001) J. Immunol. Methods. 251:123-35), Hanseula or Saccharomyces.

In one preferred embodiment, antibodies are produced in mammalian cells.Preferred mammalian host cells for expressing the clone antibodies orantigen-binding fragments thereof include Chinese Hamster Ovary (CHOcells) (including dhfr− CHO cells, described in Urlaub and Chasin((1980) Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFRselectable marker, e.g. as described in Kaufman and Sharp ((1982) Mol.Biol. 159:601-621), lymphocytic cell lines, e.g. NS0 myeloma cells andSP2 cells, COS cells and a cell from a transgenic animal, e.g. atransgenic mammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the diversifiedimmunoglobulin domain, the recombinant expression vectors may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g. origins of replication) and selectable markergenes. The selectable marker gene facilitates selection of host cellsinto which the vector has been introduced (see, e.g. U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr− host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

In an exemplary system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy and the antibodylight chain is introduced into dhfr⁻ CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g. derived from SV40,CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoterregulatory element or an SV40 enhancer/AdMLP promoter regulatoryelement) to drive high levels of transcription of the genes. Therecombinant expression vector also carried a DHFR gene, which allows forselection of CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium. For example, some antibodies can be isolated by affinitychromatography with a Protein A or Protein G.

For antibodies that include an Fc domain, the antibody production systempreferably synthesises antibodies in which the Fc region isglycosylated. For example, the Fc domain of IgG molecule is glycosylatedat asparagine 297 in the CH2 domain. This asparagine is the site formodification with biantennary-type oligosaccharides. It has beendemonstrated that this glycosylation is required for effector functionsmediated by Fc receptors and complement C1q (Burton and Woof (1992) Adv.Immunol. 51:1-84; Jefferis et al (1998) Immunol. Rev. 163:59-76). In onepreferred embodiment, the Fc domain is produced in a mammalianexpression system that appropriately glycosylates the residuecorresponding to asparagine 297. The Fc domain can also include othereukaryotic post-translational modifications.

Antibodies can also be produced by a transgenic animal. For example,U.S. Pat. No. 5,849,992 describes a method of expressing an antibody inthe mammary gland of a transgenic mammal. A transgene is constructedthat includes a milk-specific promoter and nucleic acids encoding theantibody of interest and a signal sequence for secretion. The milkproduced by females of such transgenic mammals includes,secreted-therein, the antibody of interest. The antibody can be purifiedfrom the milk, or for some applications, used directly.

An ApoE-CTD antibody of the invention may be isolated from the displaylibrary and its sequence and/or structure may be analysed. The antibodymay be produced in any desired quantity using known methods. Forexample, the antibody may advantageously be produced by a chemicalsynthesis followed by treatment under oxidising conditions appropriateto obtain the native conformation, i.e., the correct disulfide bondlinkages. Synthesis may be carried out by methodologies well known tothose skilled in the art (see, Kelley et al., in Genetic EngineeringPrinciples and Methods, (Setlow, J. K., ed.), Plenum Press, NY., (1990)vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis (1989),W.H. Freeman Co., San Francisco). Polypeptides according to theinvention may also be prepared commercially by companies providingpolypeptide synthesis as a service (e.g., BACHEM Bioscience, Inc., Kingof Prussia, Pa.; Quality Controlled Biochemicals, Inc., Hoplinton,Mass.).

D. Diagnostic Methods

Antibodies that bind to ApoE-CTD and identified by the methods describedherein and/or detailed herein have in vitro and in vivo diagnostic,therapeutic and prophylactic utilities.

In one aspect, the present invention provides a diagnostic method fordetecting the presence ApoE-CTD in vitro (e.g., a biological sample,such as a biopsy or in vivo (e.g., in vivo imaging in a subject).

The method includes: (i) contacting a sample with an antibody of theinvention; and (ii) detecting formation of a complex between theantibody and the sample. The method can also include contacting areference sample (e.g., a control sample) with the antibody, anddetermining the extent of formation of the complex between the antibodyand the sample relative to the same for the reference sample. A change,e.g., a statistically significant change, in the formation of thecomplex in the sample or subject relative to the control sample orsubject can be indicative of the presence of ApoE-CTD in the sample.

Another method includes: (i) administering an antibody of the inventionto a subject; and (ii) detecting formation of a complex between theantibody and the subject. The detection step can include determininglocation or time of formation of the complex.

The antibody ligand can be directly or indirectly labelled with adetectable substance to facilitate detection of the bound or unboundantibody. Suitable detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials andradioactive materials.

Complex formation between an antibody of the invention and ApoE-CTD canbe detected by measuring or visualizing either the antibody bound to theApoE-CTD or unbound antibody. Conventional detection assays can be used,e.g., an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay(RIA) or tissue immunohistochemistry. Further to labelling the antibody,the presence of ApoE-CTD can be assayed in a sample by a competitionimmunoassay utilising standards labelled with a detectable substance andan unlabelled antibody. In one example of this assay, the biologicalsample, the labelled standards and the antibody are combined and theamount of labelled standard bound to the unlabeled ligand is determined.The amount of ApoE-CTD in the sample is inversely proportional to theamount of labelled standard bound to antibody.

Fluorophore and chromophore labelled antibodies can be prepared. Sinceantibodies absorb light having wavelengths up to about 310 nm, thefluorescent moieties should be selected to have substantial absorptionat wavelengths above 310 nm and preferably above 400 nm. A variety ofsuitable fluoresces and chromophores are described by Stryer (1968)Science 162:526 and Brand, L. et al. (1972) Annual Review ofBiochemistry 41:843-868. The antibodies can be labelled with fluorescentchromophore groups by conventional procedures such as those disclosed inU.S. Pat. Nos. 3,940,475, 4,289,747 and 4,376,110. One group offluorescers having a number of the desirable properties described aboveis the xanthene dyes, which include the fluoresceins and rhodamines.Another group of fluorescent compounds are the naphylamines. Oncelabelled with a fluorophore or chromophore, the antibody can be used todetect the presence or localisation of the ApoE-CTD in a sample, e.g.,using fluorescent microscopy (such as confocal or deconvolutionmicroscopy).

Immunohistochemistry can be performed using the antibodies describedherein. For example, the antibody can synthesised with a label (such asa purification or epitope tag), or can be detectably labelled, e.g., byconjugating a label or label-binding group. For example, a chelator canbe attached to the antibody. The antibody is then contacted to ahistological preparation, e.g., a fixed section of tissue that is on amicroscope slide. After an incubation for binding, the preparation iswashed to remove unbound antibody. The preparation is then analysed,e.g., using microscopy, to identify if the antibody bound to thepreparation.

Of course, the antibody can be unlabelled at the time of binding. Afterbinding and washing, the antibody is labelled in order to render itdetectable.

The antibody can also be immobilised on a protein array. The proteinarray can be used as a diagnostic tool, e.g., to screen medical samples(such as isolated cells, blood, sera, biopsies, and the like). Ofcourse, the protein array can also include other ligands, e.g., thatbind to the ApoE-CTD.

Methods of producing polypeptide arrays are described, e.g., in De Wildtet al. (2000) Nature Biotech. 18:989-994; Lueking et al. (1999) Anal.Biochem. 270:103-111; Ge (2000) Nuc. Acids Res. 28:e3; MacBeath andSchreiber (2000) Science 289:1760-1763; WO 01/40803 and WO 99/51773A1.Polypeptides for the array can be spotted at high speed, e.g., usingcommercially available robotic apparati, e.g., from Genetic MicroSystemsand Affymetrix (Santa Clara, Calif., USA) or BioRobotics (Cambridge,UK). The array substrate can be, for example, nitrocellulose, plastic,glass, e.g., surface-modified glass. The array can also include a porousmatrix, e.g., acrylamide, agarose or another polymer.

For example, the array can be an array of antibodies, e.g., as describedin De Wildt, supra. Cells that produce the polypeptide ligands cm begrown on a filter in an arrayed format. Polypeptide production isinduced, and the expressed polypeptides are immobilised to the filter atthe location of the cell.

An antibody array can be contacted with a labelled target to determinethe extent of binding of the target to each immobilised antibody fromthe diversity strand library. If the target is unlabeled, a sandwichmethod can be used, e.g., using a labelled probed, to detect binding ofthe unlabeled target.

Information about the extent of binding at each address of the array canbe stored as a profile, e.g., in a computer database. The antibody arraycan be produced in replicates and used to compare binding profiles,e.g., of a target and a non-target. Thus, antibody arrays can be used toidentify individual members of the diversity strand library that havedesired binding properties with respect to one or more molecules.

In still another embodiment, the invention provides a method fordetecting the presence of a ApoE-CTD containing plaque in vivo. Themethod includes (i) administering to a subject (e.g., a patient havingAlzheimer's disease or systemic amyloidosis) an antibody of theinvention, conjugated to a detectable marker; (ii) exposing the subjectto a means for detecting said detectable marker bound to the ApoE-CTDcontaining plaque. For example, the subject is imaged, e.g., by NMR orother tomographic means.

Examples of labels useful for diagnostic imaging in accordance with thepresent invention include radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I,^(99m)Tc, ³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such asfluorescein and rhodamine, nuclear magnetic resonance active labels,positron emitting isotopes detectable by a positron emission tomography(“PET”) scanner, chemiluminescers such as luciferin and enzymaticmarkers such as peroxidase or phosphatase. Short-range radiationemitters, such as isotopes detectable by short-range detector probes canalso be employed. The polypeptide ligand can be labelled with suchreagents using known techniques. Foe example, see Wensel and Meares(1983) Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. fortechniques relating to the radiolabel of antibodies and D. Colcher etal. (1986) Methods Enzymol. 121:802-816.

A radiolabel ligand of this invention can also be used for in vitrodiagnostic tests. The specific activity of a isotopically-labelledligand depends upon the half-life, the isotopic purity of theradioactive label, and how the label is incorporated into antibody.

Procedures for labelling polypeptides with the radioactive isotopes(such as ¹⁴C, ³H, ³⁵S, ¹²⁵I, ³²P, ¹³¹I) are generally known. Forexample, tritium labelling procedures are described in U.S. Pat. No.4,302,438. Iodinating, tritium labelling, and ³⁵S labelling procedures,e.g., as adapted for murine monoclonal antibodies, are described, e.g.,by Goding, J. W. (Monoclonal Antibodies: Principles And Practice:Production And Application Of Monoclonal Antibodies In Cell Biology,Biochemistry, And Immunology 2nd ed., London, Orlando, Academic Press(1986) polypeptide. 124-126) and the references cited therein. Otherprocedures for iodinating polypeptides, such as antibodies, aredescribed by Hunter and Greenwood (1962) Nature 144:945, David et al.(1974) Biochemistry 13:1014-1021, and U.S. Pat. Nos. 3,867,517 and4,376,110. Radiolabelling elements which are useful in imaging include¹²³I, ¹³¹I, ¹¹¹In, and ^(99m)Tc, for example. Procedures for iodinatingantibodies are described by Greenwood, F. et al. (1963) Biochem. J.89:114-123; Marchalonis, J. (1969) Biochem. J. 113:299-305; andMorrison, M. et al. (1971) Immunochemistry 8:289-297. Procedures for^(99m)Tc-labeling are described by Rhodes, B. et al. in Burchiel, S. etal. (eds.), Tumor Imaging: The Radioimmunichemical Detection of Cancer,New York: Masson 111-123 (1982) and the references cited therein.Procedures suitable for ¹¹¹In-labeling antibodies are described byHnatowich, D. J. et al. (1983) J. Immun. Methods 65:147-157, Hnatowich,D. et al. (1984) J. Applied Radiation 35:554-557 and Buckley, RG. et al.(1984) F. E. B. S. Lett. 66:202-204.

In the case of a radiolabelled antibody, the antibody is administered tothe patient, is localised to the plaque with which the antibody reacts,and is detected or “imaged” in vivo using known techniques such asradionuclear scanning using e.g., a gamma camera or emission tomography.Alternatively, a position emission transaxial tomography scanner, suchas designated Pet VI located at Brookhaven National Laboratory, can beused where the radiolabel emits positrons (e.g., ¹¹C, ¹⁸F, ¹⁵O, and¹³N).

Magnetic Resonance Imaging (MRI) uses NMR to visualise internal featuresof a living subject, and is useful for prognosis, diagnosis, treatment,and surgery. MRI can be used without radioactive tracer compounds forobvious benefit. Some MRI techniques are summarised in publishedEuropean patent application EP-A-0 502 814. Generally, the differencesrelated to relaxation time constants T1 and T2 of water protons indifferent environments is used to generate an image. However, thesedifferences can be insufficient to provide sharp high resolution images.

The differences in these relation time constants can be enhanced bycontrast agents. Examples of such contrast agents include a number ofmagnetic agents paramagnetic agents (which primarily alter T1) andferromagnetic or superaramagnetic (which primarily alter T2 response).Chelates (e.g., EDTA, DTPA and NTS chelates) can be used to attach (andreduce toxicity) of some paramagnetic substances (e.g., Fe⁺³, Mf⁺²,Gd⁺³). Other agents can be in the form of particles, e.g., less than 10μm to about 10 nM in diameter). Particles can have ferromagnetic,antiferromagnetic or superparamagnetic properties. Particles caninclude, e.g., magnetic (Fe₃O₄),—Fe₂O₃, ferrites and other magneticmineral compounds of transition elements. Magnetic particles may includeone or more magnetic crystals with and without nonmagnetic material. Thenonmagnetic material can include synthetic or natural polymers such assepharose, dextran, dextrin, starch and the like.

Antibodies of the invention can also be labelled with an indicatinggroup containing of the NMR-active ¹⁹F atom, or a plurality of suchatoms inasmuch as (i) substantially all of naturally abundant fluorineatoms are the ¹⁹F isotope and, thus, substantially allfluorine-containing compounds are NMR-active; (ii) any chemically activepolyfluorinated compounds such as trifluoracetic anhydride arecommercially available at relatively low cost, and (iii) manyfluorinated compounds have been found medically acceptable for use inhumans such as the perfluorinated polyethers utilised to carry oxygen ashemoglobin replacements. After permitting such time for incubation, aMRI scan is carried out using an apparatus such as one of thosedescribed by Pykett (1982) Scientific American 246:78-88.

Also within the scope of the invention are kits comprising an antibodyof the invention and instructions for diagnostic use, e.g., the use ofthe antibody to detect ApoE-CTD, in vitro, e.g., in a sample, e.g., abiopsy from a patient having systemic amyloidosis, or in vivo, e.g., byimaging a subject. The kit can further contain a least one additionalreagent, such as a label or additional diagnostic agent. For in vivo usethe antibody can be formulated as a pharmaceutical composition.

E. Therapeutic Methods

Polypeptides that bind to ApoE-CTD and identified by the methodsdescribed herein and/or detailed herein have therapeutic andprophylactic utilities. For example, these ligands can be administeredto cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g. invivo, to treat, prevent and/or diagnose a variety of disorders such asAlzheimer's disease or systemic amyloidosis.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of an anti-ApoE-CTD antibody, alone or incombination with, a second agent to a subject, e.g. a patient, who has adisorder (e.g. a disorder as described herein), a symptom of a disorderor a predisposition toward a disorder, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisorder, the symptoms of the disorder or the predisposition toward thedisorder.

As used herein, an amount of an anti-ApoE-CTD polypeptide effective totreat a disorder, or a “therapeutically effective amount” refers to anamount of the ligand which is effective, upon single or multiple doseadministration to a subject, or in prolonging curing, alleviating,relieving or improving a subject with a disorder as described hereinbeyond that expected in the absence of such treatment.

As used herein, an amount of an anti-ApoE-CTD polypeptide effective toprevent a disorder, or a “prophylactically effective amount” of thepolypeptide refers to an amount of an anti-ApoE-CTD polypeptide, e.g. ananti-ApoE-CTD antibody described herein, which is effective, uponsingle- or multiple-dose administration to the subject, in preventing ordelaying the occurrence of the onset or recurrence of a disorder, e.g.Alzheimer's disease.

The terms “induce”, “inhibit”, “potentiate”, “elevate”, “increase”,“decrease” or the like, e.g. which denote quantitative differencesbetween two states, refer to a difference, e.g. a statisticallysignificant difference, between the two states.

As used herein, the term “subject” is intended to include human andnon-human animals. Preferred human animals include a human patienthaving a disorder characterised by abnormal cell proliferation or celldifferentiation. The term “non-human animals” of the invention includesall vertebrates, e.g. non-mammals (such as chickens, amphibians,reptiles) and mammals, such as non-human primates, sheep, dog, cow, pig,etc.

The term “amyloid disorders” is intended to include, but not limited to,Alzheimer's disease, primary systemic amyloidosis, secondary systemicamyloidosis, senile systemic amyloidosis, familial amyloidpolyneuropathy I, familial amyloid polyneuropathy III, familialnon-neuropathic amyloidosis, hereditary cerebral amyloid angiopathy,Familial British Dementia (FBD), Haemodialysis-related amyloidosis,Familial amyloidosis (Finnish type), Familial subepithelial cornealamyloid, Type II diabetes, Hereditary renal amyloidosis, Pituitary-glandamyloidosis, Injection localized amyloidosis, Medullary carcinoma of thethyroid, Atrial amyloidosis, Familial Danish Dementia (FDD), and Downssyndrome. Related to amyloid diseases wherein amyloid fibrils aredetected, comprise, but is not limited to, Spongiform encephalopathies,Sporadic Creutzfeldt-Jakob disease, Familial Creutzfeldt-Jakob disease,Iatropic prion disorders, Variant Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker Disease (GSS), Kuru, Parkinson's disease,Huntington's disease, Familial amyotrophic lateral sclerosis (ALS), andChronic obstructive pulmonary disease.

Furthermore, amyloid conditions can be defined as disorders with amyloiddeposits in brain, medulla or other organs. An example of such disordersis Alzheimer's disease. Other dementia disorders characterized byamyloid deposits are Spongiform encephalopathies, SporadicCreutzfeldt-Jakob disease, Familial Creutzfeldt-Jakob disease, Iatropicprion disorders, Variant Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker Disease (GSS), Kuru, Parkinson's disease,Huntington's disease, Familial British Dementia, Familial DanishDementia, Down syndrome, Primary Systemic amyloidosis, such asImmunoglobulin-light-chain-related amyloidosis, Secondary Systemicamyloidosis, such as Amyloidosis related to amyloid A protein, Familialsystemic amyloidosis, such as Familial transthyretin-associatedamyloidosis, Familial apolipoprotein A-I associated amyloidosis,Familial gelsolin associated amyloidosis, Familial fibrinogen A αassociated amyloidosis, Familial lyzosome amyloidosis, Senile Systemicamyloidosis, Familial amyloid polyneuropathy I, Familial amyloidpolyneuropathy III, Familial non-neuropathic amyloidosis, Hereditarycerebral amyloid angiopathy, Haemodialysis-related amyloidosis, Familialamyloidosis, finnish type, Familial subepithelial corneal amyloid, TypeII diabetes, Hereditary renal amyloidosis, Pituitary-gland amyloidosis,Injection localized amyloidosis, Medullary carcinoma of the thyroid,Atrial amyloidosis, Chronic obstructive pulmonary disease, and Familialamyotrophic lateral sclerosis-ALS. Detailed references can be found inJames C. Sacchettini and Jeffery W. Kelly: Nature Reviews, DrugDiscovery, Vol. 1 April 2002, 267-275.

In one embodiment, the subject is a human subject. Alternatively, thesubject can be a mammal expressing an ApoE-CTD-like antigen with which apolypeptide of the invention cross-reacts. A polypeptide of theinvention can be administered to a human subject for therapeuticpurposes (discussed further below). Moreover, an anti-ApoE-CTDpolypeptide can be administered to a non-human mammal expressing theApoE-CTD-like antigen to which the polypeptide binds (e.g. a primate,pig or mouse) for veterinary purposes or as an animal model of humandisease. Regarding the latter, such animal models may be useful forevaluating the therapeutic efficacy of the polypeptide (e.g. testing ofdosages and time courses of administration).

For in vivo embodiments, the contacting step is effected in a subjectand includes administering the anti-ApoE-CTD polypeptide to the subjectunder conditions effective to permit both binding of the ligand to theplaque and the treating, e.g. the destruction of the plaque.

Methods of administering anti-ApoE-CTD polypeptides are described in“Pharmaceutical Compositions”. Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the particular drugused.

The anti-ApoE-CTD ligands can be used directly in vivo to eliminateApoE-CTD-containing plaques via natural complement-dependent cytoxicity(CDC) or antibody-dependent cellular cytotoxicity (ADCC). Thepolypeptides of the invention can include complement binding effectordomain, such as the Fc portions from IgG1, −2, or −3 or correspondingportions of IgM which bind complement. The treatment can be supplementedby the addition of complement or serum containing complement. Further,phagocytosis of plaques coated with a polypeptide of the invention canbe improved by binding of complement proteins.

Antibody-targeted amyloid plaques can be internalised by microgliathrough type A scavenger receptors (Melanie I. Brazil, Haeyong Chung,and Frederick R. Maxfield. Effects of Incorporation of Immunoglobulin Gand Complement Component C1q on Uptake and Degradation of Alzheimer'sDisease Amyloid Fibrils by Microglia J. Biol. Chem., May 2000; 275:16941-16947). Alternatively, other mechanisms independent of themicroglial Fc receptor might play a role in clearing diffuse,3D6-immunoreactive, Thio-S-negative plaques and soluble Aβ moieties(Wilcock D M, DiCarlo G, Henderson D, Jackson J, Clarke K, Ugen K E,Gordon M N, Morgan D: Intracranially administered anti-Abeta antibodiesreduce beta-amyloid deposition by mechanisms both independent of andassociated with microglial activation. J Neurosci 2003, 23:3745-3751).Consistent with this hypothesis, Fc-knockout mice also showed reductionof plaque burden after Aβ immunotherapy (Das P, Howard V, Loosbrock N,Dickson D, Murphy M P, Golde T E: Amyloid-beta immunization effectivelyreduces amyloid deposition in FcRgamma−/−knock-out mice. J Neurosci2003, 23:8532-8).

Also encompassed by the present invention is a method of killing orablating which involves using the anti-ApoE-CTD ligand for prophylaxis.For example, these materials can be used to prevent or delay developmentor progression Alzheimer's disease, systemic amyloidosis or otheramyloid disorders.

Use of the therapeutic methods of the present invention to treatAlzheimer's disease or systemic amyloidosis has a number of benefits.Since the polypeptides specifically recognise ApoE-CTD, other tissue isspared and high levels of the agent are delivered directly to the sitewhere therapy is required. Treatment in accordance with the presentinvention can be effectively monitored with clinical parameters.Alternatively, these parameters can be used to indicate when suchtreatment should be employed.

F. Pharmaceutical Compositions

In another aspect, the present invention provides compositions, e.g.pharmaceutically acceptable compositions, which include an antibody ofthe invention formulated together with a pharmaceutically acceptablecarrier. As used herein, the term “pharmaceutical compositions”encompasses labelled ligands for in vivo imaging as well as therapeuticcompositions.

As used herein, “pharmaceutically acceptable carrier” includes anyphysiologically compatible solvents, dispersion media, coatings, and thelike. Preferably, the carrier is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g. by injection or infusion). Depending on the routeof administration, the active compound, i.e. polypeptide may be coatedin a material to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-sold and solid dosage forms, such asliquid solutions (e.g. injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for administration of humans with antibodies. The preferredmode of administration is parental (e.g. intravenous, subcutaneous,intraperitoneal, intramuscular). In a preferred embodiment, theanti-ApoE-CTD polypeptide is administered by intravenous infusion orinjection. In another preferred embodiment, the anti-ApoE-CTD ligand isadministered by intramuscular or subcutaneous injection.

The phrases “parenteral administration” and administered parentally” asused herein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnod,intraspinal, epidural and intrasternal injection and infusion.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the active compound (i.e. the polypeptide) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilisation. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The antibodies of the present invention can be administered by a varietyof methods known in the art, although for many applications, thepreferred route/mode of administration is intravenous injection orinfusion. For example, for therapeutic applications, the antibody can beadministered by intravenous infusion at a rate of less than 30, 20, 10,5 or 1 mg/min to reach a dose of about 1 to 100 mg/m² such as 7 to 25mg/m². The route and/or mode of administration will vary depending uponthe desired results.

Pharmaceutical compositions can be administered with medical devicesknown in the art. For example, in a preferred embodiment, apharmaceutical composition of the invention can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880,4,790,824 or 4,596,556. Examples of well-known implants and modulesuseful in the present invention include: U.S. Pat. No. 4,487,603, whichdiscloses an implantable micro-infusion pump for dispensing medicationat a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Of course,many other such implants, delivery systems and modules are also known.

In certain embodiments, the compounds of the invention can be formulatedto ensure proper distribution in vivo. For example, the blood-brainbarrier (BBB) excludes many highly hydrophilic compounds. To ensure thatthe therapeutic compounds of the invention cross the BBB, they can beformulated, for example, in liposomes. For methods of manufacturingliposomes, see, e.g. U.S. Pat. Nos. 4,522,811, 5,374,548 and 5,399,331.The liposomes may comprise one or more moieties which are selectivelytransported into specific cells or organs, thus enhancing targeted drugdelivery (see, e.g. V. V. Ranade (1989) J. Clin. Pharmacol. 29:685).

Dosage regimens are adjusted to provide the optimum desired response(e.g. a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parental compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody of the invention is0.1-20 mg/kg, more preferably 1-10 mg/kg. The antibody can beadministered by intravenous infusion at a rate of less than 30, 20, 10,5 or 1 mg/min to reach a dose of about 1 to 100 mg/m² or about 5 to 30mg/m². For antibody fragments which have lower molecular weights than anIgG, appropriate amounts can be proportionally less. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody of the invention. The desired therapeutic resultis typically a lessening or amelioration of one or more symptom of thedisease or disorder from which the individual being treated issuffering. A therapeutic amount of an antibody of the invention may bean amount which serves to slow down or stop production of amyloiddeposits, eliminate existing amyloid deposits, alleviate underlyingdisorders (that give rise to secondary amyloidosis), and relievesymptoms caused by heart or kidney damage. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the composition may vary accordingto factors such as the disease state, age, sex and weight of theindividual, and the ability of the polypeptide ligand to elicit adesired response in the individual. A therapeutically effective amountis also one is which any toxic or detrimental effects of the compositionis outweighed by the therapeutically beneficial effects. A“therapeutically effective dosage” preferably inhibits a measurableparameter, e.g. plaque formation or growth rate by at least about 20%,more preferably by at least about 40%, even more preferably by at leastabout 60%, and still more preferably by at least about 80% relative tountreated subjects. The ability of a compound to inhibit a measurableparameter can be evaluated in an animal model system predictive ofefficacy in humans. Alternatively, this property of a composition can beevaluated by examining the ability of the compound to inhibit suchinhibition in vitro by assays known to the skilled practitioner.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. The desired prophylatic result is the inhibition ordelay in the outset or progression of symptoms associated with thedisease it is intended to prevent in the individual being treated.Typically, since a prophylactic dose is used in subject prior to or atan earlier stage of disease, the prophylactically effective amount willbe less than the therapeutically effective amount.

Also within the scope of the invention are kits comprising an antibodyof the invention and instructions for use, e.g. treatment, prophylacticor diagnostic use. In one embodiment, the instructions for diagnosticapplications include the use of the antibody to detect the form ApoE-CTDassociated with plaques, in vitro, e.g. in a sample, e.g. a biopsy orcells from a patient having Alzheimer's disease or systemic amyloidosis,or in vivo. In another embodiment, the instructions for therapeuticapplications include suggested dosages and/or modes of administration ina patient with Alzheimer's disease or systemic amyloidosis. The kit canfurther contain at least one additional reagent, such as diagnostic ortherapeutic agent.

The following invention is further illustrated by the followingExamples, which should not be construed as further limiting. Thecontents of all references, pending patent applications and publishedpatents, cited throughout this application are hereby expresslyincorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

EXAMPLES Example 1 Antibody Library Composition

Antibodies that bind to ApoE found in plaques of amyloid disorders thatdo not bind to VLDL were selected from human phage antibodies in theDyax phagemid library Fab300. Antibody diversity is present in thelibrary used for the selections on CTD, the diversity in the light andheavy chains are composed as follows:

Heavy Chains

The heavy chain consists of one heavy chain gene segment (V3-23, orDP-47), in which diversity is created using synthetic oligonucleotidesin certain positions in the HCDR1 and HCDR2. The distribution pattern isbased on a diversity analysis of natural sequences. The appended HCDR3diversity is derived from natural occurring sequences of the IgM-pool ofB-cells from a series of autoimmune donors.

Light Chains

The light chain repertoire is derived from a pool of naturallyrearranged light chains sequences, from the same source as the H-CDR3diversity. This means that we can expect Vkappa and Vlambda genes, basedon a diverse range of germine segments and with somatic mutations in oroutside the CDRs.

Control Antibodies

The following anti-ApoE antibodies were used as controls:

3D12, a mouse antibody that binds to CTD, VLDL, LDL, ApoE2, ApoE3 andApoE4 (Colabek et al. Biophysical J., 79:1008-1015);

E19, a goat antibody directed to CTD (Weisgraber (1986), J. Biol. Chem.,261: 2068-2076); and

6C5, a mouse antibody directed to NTD (Castano 1995) J. Biol. Chem.,270: 17610-17615.

Example 2 Preparation and Pretesting of Fibrils

Fibrils were extracted from spleen and kidney. Insoluble amyloid fibrilswere extracted from human tissues by repeated rounds of mechanicalhomogenisation in cold 0.15M NaCl, 0.1% NaN3, with subsequentcentrifugation in order to rescue the amyloid in the pellet. Finally theamyloid was dissolved/suspended in water and stored. Amyloid content wasverified by Congo red staining of suspension smears. This method ofextracting fibrils is known in the art, see for example Skinner et al.Prep. Biochem. 1982;12(5): 461-476. It was determined that bound andnon-bound phage could be separated by washing with 5 Marvel-PBS-Tween(0.1%) washes, 2 PBS-Tween washes and 1 PBS wash.

Example 3 Preparation and Pretesting of Biotinylated CTD (bCTD)

CTD having the amino acid sequence shown in SEQ ID NO: 1 was used.Biotinylation was performed with sulfo-NHS-SS-biotin according to themethod described by Pierce using molar ratios of CTD/biotin of 1/2 and1/10. SDS PAGE revealed that 100% of the material was labelled. Inmass-spectrometry at a ratio of 1/10, three to five of the five possiblebiotinylation sites were labelled with biotin. At a ratio of 1/2 bCTDshowed one or two biotins per molecule which is favourable for keepingthe structure of the molecule. All CTD and NTD used were labelled withthe same protocol at a ratio 1/2 and tested in SDS-page.

Coated bCTD was prepared by coating BSA with biotin, washing, addingstreptavidin and, after washing again, adding b-CTD.

To produce denatured bCTD, bCTD was treated with urea and, afterwashing, bound to streptavidin on biotinylated BSA. To be sure that thebCTD binds and stays on the beads during urea treatment, we measured theamount of bCTD before and after urea treatment and binding to the beads.No loss of bCTD was seen.

Example 4 Selections on Fibrils and ur-bCTD

In the selection strategy the aim was to select for antibodies bindingto the complex form of CTD in amnyloid fibrils. A first round ofselection was performed on fibrils from kidney, a second round ofselection was performed on fibrils from spleen and rounds three and fourof selection were performed on ur-bCTD. No enrichment was found inrounds two and three but in round four there was an enrichment of 1379.In a second strategy, three rounds of selections on bCTD were performed.

A pre-screening was then carried out to decide which round of selectionto chose for a high throughput screen and which antigen should be used.There was no significant difference seen between screening on bCTD andur-bCTD. It was, therefore, decided to carry out a high throughputscreen on the ur-bCTD selection with streptavidin coated bCTD. As anegative control bBSA, streptavidin coated plates were used.

Example 5 Sequence and Binding Analysis

Screening of more than 2000 clones was performed on coated bCTD. ELISAwas performed in an automated system. The same method was used to screenfor NTD binders. Clones, binding to bCTD and not binding to bNTD wereanalysed further.

Sequencing was carried out for heavy and light chains genes for clonespositive in the high throughput bCTD phage ELISA. Dyax's proprietary‘Webphage’ software was used to analyse the sequences of clones.

752 clones were screened after round 3 (23 positives) and 216 clonesafter round 4 (216 positives) of the selections on fibrils and ur-bCTD.940 clones were screened after round 2 of selections on bCTD. The numberof positive clones in ELISA using bCTD was 463. The number of correctsequences obtained was 163.

To analyse the sequence data, comparisons of sequences at differentlevels were carried out, including the following:

(1) overall diversity=number of different sequences, will identify aclone as different if one amino acid difference is found (dubbed VH+VLdiversity)=163

(2) heavy chain diversity=number of clones with a different heavy chain,ignoring the light chain sequence=152

(3) HCDR3 diversity=the number of clones with a different heavy chainCDR3=54 (+2 clones with amber HCDR3).

This three-level analysis was carried out for two reasons:

Firstly, it is generally accepted that the heavy chain, and, in thatdomain, its HCDR3 region, are of major importance for the epitoperecognition of the antibody. All antibodies with an identical H-CDR3sequence were grouped.

Secondly, the design of the diversity of the library is such thatantibody variants that have an identical HCDR3 with mutations in theother CDRs of the heavy chain, and sometimes identical, sometimesdifferent light chains are expected.

Example 6 Binding of Antibodies to bCTD and VLDL

To monitor binding to VLDL, VLDL was coated on a microtiter plate, andincubated with the test antibodies. A secondary antibody-HRP detectionmethod is used to detect bound antibodies. Staining is performed withtetramethylbenzidine (TMB) and H₂O₂. Only non-bound antibody is washedaway. Binding to VLDL will give a high signal in ELISA.

As a positive control we used monoclonal antibodies against ApoE. Twoantibodies (3D12, E19), binding to CTD and VLDL, were positive in bCTDELISA (FIG. 1A) and in VLDL ELISA (FIG. 1B). Another antibody (6C5) thatbinds to NTD does not bind to bCTD but binds well to VLDL. This NTD siteis not covered by VLDL and could give a measure of coating quality ofApoE itself. Since of this antibody signal is high, we can conclude thatenough ApoE is coated to perform the VLDL ELISA like it is.

For phage antibodies (FIG. 2), we made a classification in 3 groups:antibodies that are always positive (more than 3 times of negative phagebinding), antibodies that are sometimes positive, sometimes negative(sometimes 2 times negative phage binding, sometimes negative) andnegative antibodies. For the doubtful antibodies the ELISA is possiblynot sensitive enough, or the antibodies are just not binding or probablythe affinity is not high enough to see a high signal or there could be across-reaction with VLDL (epitope partially on VLDL, partially oncovered CTD) etc.

Example 7 VLDL Assay Development and Automation

The VLDL ELISA was performed for all 203 bCTD positive phage clones. 6clones were found which were always positive (more than 3 times thebackground). Other clones produced a signal higher than 2 times thebackground (FIG. 3). These clones are not excluded from further testingat this stage. Only 6 clones were positive, with a high signal, to VLDL.Tests were carried out 3 times, and the same results were obtained. Inparallel binding of antibodies to bNTD was tested. No antibodies boundto bNTD. Clones that bound to VLDL were not tested further.

Example 8 Recloning of phase to Fab, Specificity Tests

Because of the low amount of VLDL binders in coated VLDL ELISA andbecause of the variable results of the VLDL competition ELISA, we batchrecloned in parallel all different 157 clones from Fab on phage intosoluble Fab. It was expected that many Fabs will not bind to bCTDbecause of their monovalent nature versus the multivalent phage, thusenabling low affinity binders to be excluded by means of Fab ELISAsignals on bCTD.

After recloning, 85 antibodies bound specifically to bCTD. No new VLDLor NTD binders were found. The amino acid sequences of CDR regions ofthe VH and VL chains of these antibodies are shown in Tables 9 and 10.

Example 9 Epitope Mapping

Binding to identical epitopes is tested by monitoring competitionbetween Fab and phage antibodies. A limited amount of phage and amaximal amount of Fab is added to an ELISA well coated with bCTD. Afterbinding steps, phage is detected by a peroxidase reaction afterincubating with an anti-M13 HRP antibody. Because of the highconcentration of Fab added, phage directed to the same epitope as theFab will be competed off and the Fab signal will be decreased.

Antibodies from the same VH-CDR3 group recognise overlapping epitopes.This criterion was used to exclude clones for immunohistochemistry(IHC): Only clones with the highest/slowest off-rate were tested. Allclones not belonging to a big VH-CDR3 group were tested in IHC.

Antibodies 807A-M0026-F05 and 807A-M0027-E11 did not cross-react witheach other. However, both cross-reacted with antibody 807A-M0028-A07,indicating that both antibodies recognise a similar but not the sameepitope.

Antibody 807A-M0028-B02 possibly recognises another epitope thanantibodies 807A-M0026-F05 and antibody 807A-M0027-E11.

Example 10 Off-Rate Measurements

To optimise Biacore measurements, we used a Biacore chip coated withstreptavidin to bind bCTD. First we analysed antibodies 3D12 and E19 forbinding to the chip. We also recloned a Fab that we recovered from thepre-screening (not binding to VLDL, positive in Fab ELISA). Mab 3D12 didnot bind in Biacore, probably due to low affinity. Ab E19 andnon-purified Fab 1F7 (dialysed periplasmic fraction) did bind to thebCTD chip. As a control we used a channel coated with bBSA; neitherantibody bound to this surface.

Off-rate ranking of selected Fabs was determined with this bCTD chipusing periplasmic extracts (important for ranking clones, now and duringaffinity maturation studies). Table 1 shows a representative list ofoff-rates.

Affinity determination was determined on a low density of bCTD on thechip using purified Fab fragments.

Example 11 Immunohistochemistry

Immunohistochemistry (IHC) on the antibodies with the slowest off-rateof the identical VH-CDR3 groups and all antibodies having a differentVH-CDR3 (single clones) were tested in IHC performed on frozen tissueslides.

For IHC and affinity measurement purified Fabs were used. Antibodyfragments (Fabs) were expressed in bacteria (typically in 400-mlcultures, 4 hours IPTG induction) and purified from periplasmic extractsby Immobilized Metal Affinity Chromatography (IMAC). Periplasmicextracts were prepared by “osmotic shock” treatment of the bacteria. Thesamples were loaded on a 1 ml Co-IDA column and eluted with a 0-150 mMlinear gradient of imidazole. Protein preparations were dialyzed againstPBS and analyzed by non-reducing SDS-PAGE.

Only 3 antibodies bound as Fab on AD plaques: 807A-M0027-E11,807A-M0028-B02 and 807A-M0026-F05.

Antibody 807A-M0027-E11 detects Alzheimer's Disease (AD) plaques in IHC.It is important that antibodies specifically recognise plaques in tissueand not to Apolipoproten E exposed in serum. Typically, an antibody ofthe invention binds to plaques of at least two patients having AD and topatients potentially also having systemic amyloidosis. Therefore, IHCwas performed in the presence of fresh plasma. In IHC staining with807A-M0027-Eli, as phage and as Fab, in the presence of even 50% plasma,the signal was not quenched. Also the addition of VLDL in solution didnot change the staining. This is in contrast with the 6C5 controlantibody, directed to NTD, the signal of which was quenched by freshplasma or VLDL solution. SFab antibody 807A-M0028-B02 stained positiveon AD plaques and also astrocytes. SFab antibody 807A-M0026-F05 stainedAD plaques weakly. The staining pattern for this antibody is not verystrong, caused by low affinity of this antibody. The antibodies werepositive in tissues of more than 1 patient.

Other sFab antibodies: 807A-M0039-C10, 807A-M0037-D01, 807A-M0046-A06and 807A-M0039-C10 only detected astrocytes on AD brain tissue.

Example 12 Affinity Measurement of sFabs Binding to Plagues in IHC

The three Fabs 807A-M0027-E11, 807A-M0028-B02 and 807A-M0026-F05 wereextensively studied in Biacore analysis. First bCTD was coated on astreptavidin chip, then sFabs were run over the chip at differentconcentrations and binding resonance units (RU) were measured. As anegative control, one channel of the chip was saturated with biotin-BSA.FIG. 4A shows the analyses of sFab antibody 807A-M0027-E11 on bCTDresulting in an affinity is 47.8 nM. The affinity of sFab antibody807A-M0028-B02 showed a similar pattern, with an affinity of 179 nM(FIG. 4B). Antibody 807A-M0026-F05 has such a low affinity (in the μMrange) that it is difficult to measure (FIG. 4C).

In contrast, when sFabs were coupled to a CM5 chip via an anti-Fcantibody, no binding was observed for antibody 807A-M0027-E11 (see FIG.4D), nor for antibody 807A-M0028-B02.

The different results reversing the coating and analyte, in the twodifferent Biacore measurements suggest that both, antibody807A-M0027-E11 and 807A-M0028-B02 only bind to coated bCTD and not tobCTD in solution.

Example 13 Antibody Reformatting Expression and Purification of IgGs

Antibody (Batch) Reformatting to IgG1

85 clones showed specific binding to CTD as soluble Fab. Of these the 30candidates that had been chosen for IHC studies were reformatted tocomplete human IgG1 antibodies.

A pool of 157 CTD-specific Fabs, that contained all 85 “soluble Fabbinders”, was used for simultaneous, restriction digestion basedbatch-reformatting into the human IgG1 expression vector pBh1.

The batch-reformatting strategy involved two cloning steps, and isillustrated in FIG. 5. In the first step, complete Fab fragments areinserted into pBh1. In the second step, internal/regulatory sequencesare exchanged.

To “re-identify” the initial Fabs, about 300 individual clones wereanalysed by DNA-sequencing. 72 of the 85 “soluble Fab binders” werefound back. Notably, 29 of 30 of the prioritised candidates chosen forIHC (as phage and soluble Fab) were obtained as IgG1 constructs by batchreformatting.

11 of the 13 remaining “soluble Fab binders” could be reformattedindividually into the human IgG1 expression vector pRh1. Identity of thereformatted antibodies to their Fab counterparts was verified bysequencing. Besides initial PCR-amplification of the completeFab-insert, the cloning approach is identical with thebatch-reformiatting strategy depicted in FIG. 5.

Expression and Purification of IgG1 Antibodies

Reformatted IgG1-antibodies were expressed in transiently transfectedHEK293T cells. Antibodies were purified from culture supernatant of˜5×10⁶ transfected cells (per flask), kept in culture for about oneweek. Purification was carried out by Protein-A-based affinitychromatography. Purified antibodies were dialyzed against PBS andanalysed on SDS-gel under reducing and non-reducing conditions.

Biotinylation of IgG Antibodies

Biotinylation of antibodies was performed in PBS, incubating thepurified antibodies for 2 hours with a 15-fold molar excess ofSulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. The levelof biotin-incorporation (i.e. the average number of biotin groups perantibody molecule) was determined using the HABA[2-(4′-hydroxyazobenzene) benzoic acid] method (Pierce). Using thisapproach, we found that all biotinylated antibodies contained 3 to 4biotin groups per molecule.

Example 14 Specificity Tests with IgG1 Antibodies

Binding of IgG1 Antibodies to Other Species

Cross-reactivity of the human antibodies on bCTD of these species wastested to try to identify antibodies that can be studied in mouse andprimate models.

The antibodies (807A-M0028-B02, 807A-M0027-E11, 807A-M0026-F05) thatbound to plaques in AD tissues as sFab, were reformatted to hIgG1 andtested for their binding capacity on recombinant mouse CTD (mbCTD),recombinant primate CTD (pbCTD) and recombinant human CTD (hbCTD) thatwas biotinylated.

Antibodies 807A-M0028-B02 and 807A-M0027-E11 (FIGS. 6A and 6C) did bindto CTD of the three different species. Both antibodies bound to the sameextent suggesting that the epitopes recognised by the antibodies are thesame in these species.

Antibody 807A-M0026-F05 (FIG. 6B) binds to pCTD and hCTD but not tomouse CTD, suggesting that the antibody is directed against an epitopethat is not present in mice or that concentration used is not highenough. The low O.D. in this ELISA can be explained by the low affinityof this antibody.

Binding of IgG1 Antibodies to bNTD

807A-M0027-E11 and 807A-M0026-F05 did not bind to bNTD. Antibody807A-M0028-B02 did bind at very high concentrations (10 and 5 μg/ml). Tocheck if this was related to specific binding, we tried to compete with1000 times more bNTD in solution (540 ug/ml in solution, 0.5 ug/mlcoated) (FIG. 7). The non-specific signal did not decrease incompetition. The signal is most probably due to the high amounts ofantibody added, as also seen for the anti-MUC1 antibody PH1 (FIG. 7).

Binding of IgG1 Antibodies to VLDL

Binding of antibodies to coated VLDL was tested in ELISA. Unlike thephage and the Fabs of the plaque binders, antibodies 807A-M0028-B02(FIG. 8) and 807A-M0027-E11 are binding to VLDL to the same extent. Whencompared to the binding of a non-CTD binder (PH 1, which is a MUC 1binder) this binding seems to be specific.

Because VLDL is composed of ApoE and a lipid, the coated VLDL couldchange its conformation during treatment in ELISA and it could be thatthe CTD is not covered by the lipid anymore. Therefore we did acompetition test with the antibodies in solution, bCTD bound to theplate and an excessive amount of CTD or VLDL in solution (FIG. 9). Inthis assay we could not see inhibition with VLDL for the humanantibodies (FIGS. 9A and B) and little inhibition with CTD for antibody807A-M0028-B02 (FIG. 9B). In contrast, the commercial antibodies 3D12and E19 directed against CTD were clearly inhibited by VLDL as well asby CTD. The 6C5 monoclonal directed to NTD did not bind in this assay.

These results suggest that the human antibodies (807A-M0028-B02,807A-M0027-E11) are not binding to VLDL in solution and recognise CTD insolution to a lesser extent than coated bCTD.

Conclusion

Two of the three antibodies binding to plaques in AD (807A-M0028-B02,807A-M0027-E11) are cross-reactive with pCTD and mCTD. The thirdantibody (807A-M0026-F05) does not cross-react with mCTD and binds pCTD.The antibodies of interest do not bind to NTD. Two antibodies(807A-M0028-B02, 807A-M0027-E11) bind to coated VLDL, when high antibodyconcentrations were used. Antibody 807A-M0026-F05 does not show theseproperties but this could be related to the affinity of the antibody.

Example 15 Biacore Analysis

To compare Fab and IgG1 binding and to study the nature of binding ofthe human antibodies on CTD in solution, Biacore was extensively used.

Comparison of Fabs and IgGs in Biacore

FIGS. 10, 11, 12 and 13 summarize the results of the Biacore analysis ofthe CTD-specific clones 807A-M0026-F05, 807A-M0027-E11 and807A-M0028-B02. As expected all three plaque binders bind better to bCTDon the chip as IgG1 compared with their original Fab format. IgG1Antibody 807A-M0027-E11 binds 3 times better than antibody807A-M0028-B02 while antibody 807A-M0026-F05 binds with a very low micromolar avidity to the chip.

The avidity measured (Table 2) is higher when measured 50 seconds afterthe injection of the antibody is stopped as compared with the aviditymeasured immediately after injection stop. This difference is probablydue to the rebinding of the antibody to the chip when free bCTD isavailable on the chip.

Binding of Antibodies to Captured CTD

Because of the inconsistency of VLDL ELISA in which the human IgG1antibodies bind to coated bCTD/VLDL but not CTD/VLDL in solution,additional Biacore experiments were performed (see also Biacore onFabs). In these experiments bCTD or ApoE was captured. First anti-hFcantibody was coupled to the chip, followed by binding of the specificantibodies, followed by the injection of bCTD or ApoE.

FIG. 12 shows that Fab antibody 807A-M0027-E11, indirectly coupled tothe chip, does not bind bCTD (280 nM). The same curves (data not shown)were obtained by using Fab antibody 807A-M0028-B02 and when IgG1 of bothantibodies was used. The test was performed with ApoE originating fromhuman serum. Only a very small amount of ApoE (17 RU, <1%) was bound tothe antibody (FIG. 13).

Conclusion

In Biacore, IgG1 antibodies 807A-M0027-E11 and 807A-M0028-B02 bind tocoated bCTD with nM avidity while IgG1 antibody 807A-M0026-F05 bindswith μM avidity. Affinity measurements on antigen in solution do showthat both antibodies 807A-M0027-E11 and 807A-M0028-B02 do not capturebCTD nor ApoE efficiently. These results confirm the results seen byVLDL/CTD competition ELISA: antibodies 807A-M0027-E11 and 807A-M0028-B02bind better to coated bCTD than to CTD in solution.

Example 16 Additional Testing to Study Binding of Antibodies to NaturalCTD to Peptides

SDS-PAGE Analysis of the Purified hApoE

The purified, hApoE was analyzed by reducing SDS-PAGE followed byCoomassie staining. As expected, the protein migrated as one major bandof ˜35 kDa, but there was also a broad band at ˜70 kDa and a faint smearfrom 70 kDa to ˜200 kDa. Both the 35 kDa band and the higher molecularweight species were shown by western-blot to contain hApoE.

Immunoprecipitation of Purified hApoE

Purified hApoE was immunoprecipitated with 807A-M0028-B02,807A-M0027-E11 and 807A-M0026-F05. As a positive control, we used E19, agoat anti-hApoE antibody. As a negative control, we used the PH1antibody. Untreated, purified hApoE was also included as a reference.The samples were analyzed by SDS-gel, the proteins were transferred to anitrocellulose membrane and hApoE was detected by western-blot.

As expected, the E19, the 807A-M0028-B02 and the 807A-M0027-E11antibodies were able to specifically immunoprecipitate hApoE, althoughnot very efficiently. Interestingly, the E19 antibody seemed to bespecific for the 35 kDa band, whereas the 807A-M0028-B02 and807A-M0027-E11 antibodies were more specific for the high molecularweight species. The 807A-M0026-F05 was by contrast unable toimmunoprecipitate hApoE, probably due to the low affinity of theantibody.

Immunoprecipitation of Cell Lysates

Cell lysates of PBMC were immunoprecipitated using 807A-M0028-B02,807A-M0027-E11 and 807A-M0026-F05, as well as E19 (positive control) andM43G5, M43F8, PH1, A2, herceptin and a human IgG1 Kappa Myeloma antibody(negative controls). Samples were analyzed by SDS-PAGE under reducingconditions followed either by silver staining or Western-blot.

Only E19 immunoprecipitated a band of the expected size (i.e. 35 kDa).The material immunoprecipitated with E19 also gave a very faint signalin Western-blot. This suggests that some hApoE is captured by E19 in thecell lysates, although this finding must be regarded with care since (1)807A-M0028-B02 and 807A-M0027-E11 did not immunoprecipitate any hApoEfrom the cell lysates, (2) 807A-M0026-F05, which was shown to be unableimmunoprecipitate hApoE, also gave a weak signal in Western-blot and (3)A2 and 807A-M0043-F08, two irrelevant antibodies, gave strong signals inWestern-blot.

Importantly, the three antibodies investigated here (807A-M0028-B02,807A-M0027-E11 and 807A-M0026-F05) did not seem to immunoprecipitate anymajor component of the cell lysates. The background for 807A-M0027-E11was a little bit higher, but not higher than e.g. PH1. These resultsindicate that the overall specificity of the antibodies, 807A-M0028-B02,807A-M0026-F05 and 807A-M0027-E11, is due to the binding to CTD.

Immunoprecipitations of VLDL

Binding of antibodies to VLDL was also tested by immunoprecipitation.10% VLDL was used in these tests. As detection antibody 6C5 was used.After 5 minutes development of a Western blot with the ECL method, noApoE was detected for the human antibodies of interest. After overnightdevelopment antibody 807A-M0027-E11 and 807A-M0028-B02 were detected asfaint bands when compared with the VLDL control (non-immunoprecipitatedVLDL, 10% of the amount that was used for immunoprecipitation).Immunoprecipitation with the 6C5 antibody showed a more extensive bandthan with the human antibodies.

Discussion

807A-M0028-B02 and 807A-M0027-E11, but not 807A-M0026-F05,preferentially immunoprecipitate a high molecular weight from of hApoEpurified from plasma. The nature of these species remains unclear,although it is clear that they contain hApoE and must form very stablecomplexes. These antibodies do not interact significantly with majorcellular components. Immunoprecipitation of VLDL with the antibodies ispossible, although the amount might be very low.

Example 17 In Vivo Studies

Mice were bred to express in the brain the human gene for amyloidprecursor protein (APP): Swedish mutation K670N,M671L, APP Line 2576,driven by the hamster prion promotor (Hsia et al Science 1996,274:99-102), alone or in combination with a mutated human presenilin 1(PS1):M146L driven by the platelet derived growth factor (Duff et al,Nature 1996, 383 (6602):710-3, Holcomb et al, Nature Med 1998,4:97-100).

For studies requiring expression of human ApoE mice were bred thateither expressed the human ApoE4 driven by the glial fibrillary acidicprotein (GFAP) promotor with or without the mouse ApoE gene knocked out(Sun et al, J Neurosci, 1998, 18:3261-3272) in combination withhumAPP:Swe and humPS1:M146L or humAPP:Swe only.

Monoclonal antibodies (mAb) of the human immunoglobulin G1 (hIgG1)isotype to C-terminal domain (CTD) of Apolipoprotein E (ApoE) wereinjected intra-peritoneally at a concentration of e.g. 10 mg/kg in nontransgenic or transgenic mice. Some mice were injected once andsacrificed 2 days after injection and some were injected twice, with therepeated dose injected after 2 days, and then sacrificed after anadditional 2 days i.e. 4 days after the initial injection. Theconcentration of injected antibody was monitored by ELISA to CTD bindinghIgG. Brains from mice injected with a streptavidin specific monoclonalantibody served as negative controls. The sampled brains wereimmediately frozen to −70° C. and then subjected to freeze sectioning.

Staining for presence of human IgG in brain sections showedhomogeneously stained plaques evenly spread trough cortex andhipocampus. No other brain structures showed staining after in vivoexposure. In contrast, several structures along with the amyloid plaqueswere stained after ex vivo exposure. 70% of the plaques that wereaccessible for staining ex vivo by anti-ApoE CTD antibody or with a mAbto Ab were stained after the 2-day (n=3) or 4-day (n=3) in vivo exposurewith the anti-ApoE CTD antibody. The in vivo exposure for 2 or 4 days atthe given dose did not saturate the available binding sites as indicatedby the additional staining intensity obtained after ex vivo addition ofmore anti-CTD antibody.

Antibodies, as macromolecules in general, do not pass freely over theblood brain barrier (BBB). The passage of IgG is considered to be verylimited and concentrations in CSF under 0.5% of the plasma concentrationhas been reported (Elovaara et al, 1987 Eur Neurol 26:229-34, Ganrot &Laurell 1974, Clinical Chemistry 20:571-3). Staining for presence of theintraperitoneally injected human IgG in brain sections revealed that theApoE CTD specific antibody reached the cerebral plaques evenlythroughout the different brain regions in these transgenic miceindicating sufficient BBB passage for staining by immunohistochemistry(IHC) technique. Alzheimer's disease (AD) plaques are complex structuresvarying in size and density. The cerebral amyloid plaques found in thesetransgenic mice are considered to represent the small diffuse and mediumsize plaques found in AD. The plaques were homogeneously stainedindicating that the mAb did not only reach the outer layer of theplaques but penetrated the whole plaque structure. 70% of the plaquesaccessible for staining ex vivo were stained after the in vivo exposure.Considering that the dose of 10 mg/kg administered interperitoneally didnot saturate the available plaque-binding sites, a non-saturated levelof antibodies may still result in an antibody mediated plaque breakdownby FcR bearing phagocytic cells.

Example 18 Reformatting of Antibodies to Mouse IgG2a

For in vivo testing the variable regions of antibodies 807A-M0028-B02,807A-M0027-E11, 807A-M0026-F05 and a control antibody (anti-Streptavidinclone A2) were recloned into vector that contains mouse IgG2a constantregions of the heavy chain and mouse Ckappa and the variable regions ofantibodies.

These clones were transferred from a human IgG1 expression vector (pBh1)to a construct for expression of mouse IgG2a antibodies (pRmk2a), thatbesides the constant heavy chain region, also contains the mouseconstant kappa light chain gene. The VL and VH regions were lifted fromthe human IgG1 expression plasmid via PCR, and cloned sequentially intopRmk2a. VL was inserted as an ApaL1/BsiW1 fragment, 3′ of the antibodyleader and 5′ of the constant kappa gene. In case of the VH region, the5′ adjacent IRES motif was also included in the PCR amplificationproduct; an Asc1/Nhe1 fragment was inserted in pRmk2a. Integrity of theconstructs was verified by DNA sequencing. The cloning strategy isdepicted in FIG. 14.

Example 19 Preparation of Peptides

10 peptides (length 16 amino acids) covering the full ApoE CTD weresynthesized.

The peptides contain an 8 amino acid overlap between each other as shownin FIG. 15. The peptides contain an S—S Biotin group that enablesbinding to Strepatavidin (magnetic beads selections). In addition eachpeptide contains a Cystein that can be coupled to a carrier protein(BSA).

Peptides were solubilised in dimethylformamide (DMF), and subsequentlydiluted in water. All peptides, except peptide 4 were soluble in DMF ata concentration of less then 10%. Coupling was performed in 10% DMF forall peptides, except for peptide 4 which was coupled in 30% DMF. Anexcess of maleimide-activated BSA was used to bind to the peptides.After incubation an excess of Cystein was used to occupy possible freecysteins. The BSA coupled peptides (bpeptide-BSA) were used forselections.

Example 20 Binding of Antibodies to Overlapping Peptides

Antibodies 807A-M0026-F05, 807A-M0028-B02 and 807A-M0027-E11 identifiedin Example 5 were tested for their binding to overlapping peptides. Thiswas done firstly to test which peptides could be preferentially used infurther selections: bpeptide-BSA or bpeptide and secondly to see whetherthe antibodies selected in Example 5 were binding to the overlappingpeptides and if so, whether the epitopes they recognised were differentand supported the epitope mapping by competition results as performed inExample 9. Both human antibodies and mouse antibodies were compared inthis way.

For the human antibodies, peptide mapping was performed with IgG1 aswell as with the phage displayed Fab. For example, clone 807A-M0026-F05recognised bpeptide-BSA 4 and 8 as phage Fab fragment as well as wholeIgG1. The ELISA was not sensitive enough to show binding to bpeptide 4and 8 for this antibody. Therefore, we decided that it would be best tostart the selections on bpeptide-BSA to capture the majority of peptidebinders and then, if necessary, use the bpeptide in a later round ofselection. Remarkably, antibody 807A-M0026-F05 bound better to CTD thanto peptide compared with the murine monoclonal antibodies. Antibody807A-M0028-B02 bound to peptide 4. Antibody 807A-M0027-E11 did notsignificantly bind to any of the overlapping peptides.

In Example 9 we found that the epitope recognized by antibody807A-M0026-F05 and antibody 807A-M0027-E11 was covered by a large groupof antibodies. Both antibodies did not compete with each other. Sincethe affinity for CTD for antibody 807A-M0026-F05 is very low as comparedwith antibody 807A-M0027-E11, one would expect that, if they recognisethe same epitope, antibody 807A-M0027-E11 would have bound more stronglyto peptides 4 and 8 than antibody 807A-M0026-F05. Therefore, one couldconclude that both antibodies recognise related but not identicalepitopes. Antibody 807A-M0028-B02 bound to peptide 4 and was differentfrom the antibody group of the two other antibodies in competitionepitope mapping and could recognise a different epitope.

We also tested control mouse antibodies on overlapping peptides.Antibodies 3H1, 12D10 and E19 bind to peptides 3, 10 and 5+10respectively. In contrast with the human antibodies, all controlantibodies bind to about the same extent to peptide and bCTD.

Example 21 Selections and Screening on Peptides

Three rounds of selection were carried out on 10 overlappingbiotinylated peptides conjugated to BSA (b-peptide-BSA) and one round ofselection on the corresponding biotinylated peptides (b-peptide).Selection was performed on 10 individual peptides usingStreptavidin-magnetic beads. To handle this high number of selections inbetween selection rounds no titration of input/output was performed(liquid amplification).

The procedure used is set out in FIG. 16. First, three rounds ofselection for binding to bpeptide-BSA was carried out using theautomated Kingfisher system. Pre-screening of round 2 and 3 withFab-displayed on phage showed that the frequency of positive clones waslow in round 2 and that many clones were binding to BSA in round 3,despite extensive depletion and subtraction on BSA. Most likely thebinders were directed to the linker molecules on the BSA that we usedfor coupling the peptides. Therefore another round 3 selection onbpeptides was performed. For this selection background binding wasnegligible.

To reduce sequencing efforts, phage-Fab clones were batch reformatted toproduce sFabs and the large screening of the clones (ELISA andsequencing) was performed at sFab level. 307 antibodies were foundpositive in ELISA of which 46 were unique as was determined bysequencing as shown in Tables 11 and 12.

Example 22 Selections and Screening on Fibrils and Peptides

Two rounds of selection were carried out on fibrils originating from anorgan with amyloid plaques, 2 rounds on 10 corresponding b-BSA-peptidesand 1 round on 10 corresponding b-peptides.

The procedure used is summarised in FIG. 17. Round 3 and round 4 wereperformed on individual bpeptides-BSA. After pre-screening few positiveclones were found. Therefore a fifth round of selection was carried outusing bpeptides. In a pre-screen, positive clones for peptide 4 and 8were found. For these two selections we screened sFab after batchreformatting. In total 390 sFabs were screened, 109 were positive inELISA and 4 clones were unique. The amino acid sequences of the VH andVL chains of these unique clones are shown in Tables 11 and 12. In thesestrategies most clones did show unique VH-CDR3s (Table 12), and highenrichment was found: one clone was enriched 148 times and was found instrategy B1 as well as in strategy B2. This is in contrast withselections of Example 5.

Few clones (Table 13) bound to bCTD, which makes them unique as comparedto results in Example 20 where bCTD binders bound weakly or not at allto peptides. In the selection campaigns of Examples 21 and 22 no Fabswere binding to VLDL nor to NTD.

11 Fabs were found positive in IHC, 10 of these Fabs originate from theselection campaign of Example 11 and one originates from the selectioncampaigns of both Examples 21 and 22.

Example 23 Selections and Screening on ur-bCTD and Peptides

Two rounds of selection were carried out on urea treated biotinylatedCTD (ur-bTD) followed by two rounds on fibrils originating from an organwith amyloid plaques. The procedure used is summarised in FIG. 18.

Two rounds of selection on urea-CTD antigen were performed followed by 2selection rounds on fibrils 1 from an AD patient. In this strategypre-screening was done after the 3^(rd) and 4^(th) round of selection.Frequencies were 82/95 and 83/95 respectively. During pre-screening weused ur-bCTD and bCTD and no difference was found between both types ofantigen. After the third selection round, many clones were retained thatbound to bCTD but not to fibrils while after 2 rounds of selection onthe fibrils (4^(th) round of selection) the chance that these bindersare retained was less. Therefore, large scale screening was performed onthe 4^(th) round of selection. 950 sFabs were screened on ur-bCTD. 233clones were positive in ELISA as soluble Fab, 83 were unique. The aminoacid sequences of the VH and VL chains of these uniques Fabs are shownin Tables 14 and 15. In VLDL ELISA, 5 Fabs bound were positive or hadquestionable binding.

No Fabs bound to bNTD.

Many Fabs belonged to large families of identical VH-CDR3 groups; oneindividual antibody was enriched 247 times. Testing in IHC was performedon individual clones (not belonging to a large VH-CDR3 group) and clonesbelonging to a large VH-CDR3 group that were selected for a slowoff-rate.

In a first IHC screen, 6 Fabs bound to plaques of AD patients. Theproperties of the bCTD binders are summarised in Table 17.

Example 24 Production of Candidate Clones as Soluble Fabs for IHC

Fabs were produced for further analysis. ELISAs were performed onperiplasmic fractions of 100 μl cultures. Biacore off-rate measurementon periplasmic fractions of 50 ml cultures of all clones found in tableswith Biacore results.

About 90 soluble Fab proteins were produced for testing in IHC. At least10 μg was required for initial analysis. Because of wide variation insoluble Fab expression levels, the protein had to be purified eitherfrom the peripheral extract of 50 ml bacterial cultures, or fromperipheral extracts of 400 ml cultures, by IMAC chromatography, using96-well filter plates and a vacuum manifold. The yields mainly rangedbetween 10-100 μg.

Large Scale Production of IHC Positive Soluble Fabs

15 of the candidate Fabs turned out to be positive/potentially positivein IHC on plaque tissue. Of these clones more soluble Fab protein wereprepared for additional testing.

Soluble Fab proteins were prepared from the periplasmic extracts of 400ml bacterial cultures.

Example 25 Epitope Mapping Comparison with Preliminary IHC Results

High throughput Fab screening was performed on the peptides they wereselected on and also on a non-overlapping peptide as a control. Inepitope mapping all peptide positive antibodies were screened for theirbinding reactivity to all other peptides. Table 13 contains detailedresults.

Two Fabs bound to all peptides (most likely to the BSA-linker) and wereconsidered as non-specific.

No Fabs were identified that were specific for peptide 5 and 10(although the pre-screening Fab on phage showed some positive binders).

49 different Fabs bound specifically to peptides. 39 of the specificFabs only bound to the peptide they were selected on. One Fab selectedon peptide 1 also recognised peptide 6. 3 Fabs originating from thepeptide 4 selection also bound to peptide 9 and 3 Fabs originating fromthe peptide 9 selections recognised peptide 4. Further, 3 peptidesselected on peptide 8 also bound to peptide 4. Only 9 Fabs bound also tobCTD. This suggests that most of these Fabs bind to an epitope that isnot present in the recombinant bCTD and recognise another (possiblystretched) structure that could potentially also be found in plaques.The region that covers peptide 3 and peptide 4 is recognised as a‘selection dominant epitope’, containing 39 of all 49 specific Fabs and8 of 9 bCTD binders. Interestingly, this area of ApoE is thought to beinvolved in the binding to VLDL particles.

Hypothesis Taking into Account for The IHC Screen

39 of the 49 specific Fabs bound to peptide but not to bCTD. Thissuggests that these Fabs are not likely to recognize natural Apo-Econtained in for example VLDL particles. If such a Fab would bind in IHCit could indicate that such an epitope is unique and only found onplaques and that these Fabs would be important leads for furtherinvestigation.

Indeed, preliminary IHC data shows that four of these Fabs possibly bindto tissue in AD patients. These four Fabs all bind to peptide 4 and notto the overlapping peptides, suggesting that they recognise similar(overlapping) epitopes (group 1), probably epitope containing aminoacids of LVEDMQRQ (SEQ ID NO: 12) or a secondary structure only presentin peptide 4 and plaques.

Two other Fabs, positive in IHC and selected on peptide 4, bind topeptides 4 and 9 and to a conformation that is not present or not asprevalent in bCTD. Possibly the epitope for these two antibodies includesequence MQRQWAGL (SEQ ID NO: 13, group 2).

Another Fab possibly positive in IHC, selected on peptide 9, onlyrecognises peptide 9 and not overlapping peptide nor bCTD and couldrecognise either the epitope WAGLVEKV, (SEQ ID NO: 14) or a conformationonly present in peptide 9 (group 3) and plaques.

One Fab binds to peptide 1, 6 and bCTD. This epitope (RTRDRLDE, SEQ IDNO: 15) is not predicted to be inside of the binding site of VLDL (group4).

Two antibodies, obtained from selections on peptides 4 and 9, recogniseboth peptides (epitope MQRQWAGL, SEQ ID NO: 13) and CTD and thereforeare different from Fabs of group 2 (group 5).

One antibody, selected to peptide 8, binds to peptide 4, 8 (epitopeWFEPLVED, SEQ ID NO: 16) and bCTD (group 6).

Thus, according to this hypothesis the Fabs identified in Examples 21and 22, can be divided into six different groups of Fabs that eachrecognize distinct epitopes.

Example 26 Biacore Off-Rate Analysis of the Fabs Identified in theStrategies of Examples 21 and 22

Off-rate analysis of soluble Fabs was performed on all 10 peptides.Periplasmic fractions from all unique Fab of Examples 21 and 22 weremade and tested. The results confirm the epitope mapping by ELISA.

Fabs that bind to more peptides and/or to bCTD most often show the aboutthe same off-rate for those molecules. In contrast, RU's (measure forthe amount of antibody bound) are often highest for the peptide to whichthe Fabs were selected on.

The strategies of Examples 21 and 22 did not result in theidentification of antibodies belonging to large families of identicalVH-CDR3s. Therefore, off-rate measurements were not used as a criterionfor IHC.

Example 27 Epitope Mapping of the Antibodies of Example 23 andComparison with Preliminary IHC Results

In the automated screening, Fabs were screened for their bindingreaction towards ur-bCTD and streptavidin BSA as negative control. Noantibodies bound to NTD. Table 16 contains detailed results.

In total 81 different antibodies, binding to ur-bCTD and CTD were found.5 of those antibodies bound to coated VLDL. None bound to bNTD. 20antibodies, also bound to peptide. As in the strategies of Examples 21and 22 we found a selection dominant epitope around peptides 3 and 4.

Five antibodies of the 81 bound in IHC. Only one of these antibodiesbound to peptide. This antibody binds with low RU (Biacore) to peptide 4and high RU. Interestingly, this antibody was also found using thestrategies of Examples 21 and 22. The other four antibodies could becompared with antibodies 807A-M0028-B02 and 807A-M0027-E11 of Example 5.

Example 28 Reformatting of Candidate Fabs to Human IgG1

Most of the IHC positive clones described above can be individuallyreformatted to the Dyax hIgG1 expression construct pBh1 in tworestriction endonuclease based (“cut and paste”) cloning steps (see FIG.5).

With the exception of the amber-stop containing clones, 807B-M0079-D10(807B-M0027-D08) and 807B-M0081-A11 (807B-M0081-F12), and the clone807B-M0009-C03, reformatting of Fab to IgG was carried out and the IgGstransiently expressed in Hek293T cells.

The amber-stop mutation in the CDR2 of 807B-M0079-D10 is corrected onthe “phagemid level”, before the clone is reformatted using theprocedure outlined above.

The amber-mutation at the 5′-end of VL of 807B-M0081-A11 is repairedusing a different reformatting strategy/“PCR-based reformatting to thehIgG1 expression construct pRh1”. Due to the fact that the amber-stopmutation lies within the sequence of our “CJ-kappa-lifting primer”, thestop mutation is corrected during PCR amplification of the Fab fragment.The cloning strategy of PCR/Fab fragments to pRh1 is the same as the“cut and paste” approach to pBh1.

Example 29 Conclusions

229 candidate Fabs binding to CTD were isolated from a variety ofselection procedures with Dyax' human Fab300 library. Two of theprocedures included selections on peptide (Examples 21 and 22). In theselection procedure of Example 23 the successful selection of Example 5was reversed by first selecting on ur-bCTD and then on fibrils. Also incontrast with Example 5, we did not screen phage but first performed abatch recloning from phage Fab to Fab.

Very few (five) were reactive, as Fab antibody, with coated VLDL. No Fabwas positive for bNTD.

In the strategies of Example 21 and 22, some clones were enriched and weobserved VH-CDR3 groups with few individual clones.

Fab antibodies from the strategies of Example 21 and 22 recognise aselection dominant epitope around peptides 3 and 4. In the strategy ofExample 23, the same dominant epitope is found.

In IHC screening, 15 antibodies were found positive for binding toplaques in IHC in a first screen. An overview of the characteristics ofthe Fab clones positive in the first IHC screen is shown in Table 17.

Example 30 Properties of IgG1 of the 15 Fabs Positive in IHC

The 15 Fabs which were positive in Fab-IHC were reformatted to IgG1. Ofthese antibodies, nine were found positive in IgG1-IHC.

Almost all IgG antibodies bound to peptide in peptide epitope mapping.This is probably due to the higher avidity of the IgG1 as compared tothe Fab. For the antibodies selected on peptide 4 which only bound topeptide 4 as Fab, some also bound to peptide 9 as IgG. For theantibodies originally from the screen described in Example 23, the IgGantibodies bound to peptides 4 and 8, peptide 9 or peptide 7 only oneantibody, 807B-M0083E11, did not bind to any peptide.

Human CTD, mouse CTD and primate CTD were compared to each other.Several IgG antibodies bound to bCTD of each of the three species.

Although there was no binding to coated VLDL in Fab ELISA, in IgG1 ELISAseveral but not all antibodies bound to VLDL.

All IgG antibodies showed an improved binding compared to Fabs inBiacore analysis when bCTD was coated on the chip.

The IgG1 results are summarised in Table 18.

Example 31 Effect of VLDL on Binding of 807A-M0028-B02 to CTD in AmyloidDeposits

To analyse binding of 807A-M0028-B02 to CTD in amyloid deposits inpresence of lipoprotein particles, immunohistochemistry was performed inpresence or absence of VLDL to see whether the presence of VLDL wouldlead to a decreased staining intensity of plaques.

The 807A-M0028-B02 antibody was serially diluted and mixed with VLDLprior incubation of both APP/PS1 and human AD brain sections. Noquenching of signal was observed even at the lowest antibodyconcentration (0.04 μg/ml). In contrast, a commercial antibody to CTD(3H1) was completely quenched already at a high concentration ofantibody (5 μg/ml) (FIG. 20).

Example 32 Effect of 807A-M0028-B02 Antibodies on Phagoeytic Activity ofMicroglial/Macrophate Cells

To evaluate the effects of 807A-M0028-B02 on phagocytosis activity, aphagocytosis assay of CTD immobilized on Avidin-coated FluoSpherefluorescent microspheres (Molecular Probes) was developed.

The biotinylated CTD immobilized on 1.2 μm yellow-green latexFluoSpheres® NeutrAvidin™ labeled microspheres (Molecular Probes EuropeBV, Leiden, The Netherlands) were resuspended with differentconcentrations of IgG converted clone 807A-M0028-B02 diluted in OPTI-MEMmedium supplemented with 1% BSA (Tissue culture tested, Invitrogen AB,Sweden) and 2% ITS-X (Gibco, Invitrogen AB, Sweden) serum supplement(CM) and incubated for 30 min at +4° C. THP-1 cells (10⁶ cells ml⁻¹) inCM with or without 2.0% NaN₃, the inhibitor of phagocytosis, were addedat a ratio of 1:100 (cells:beads). To allow binding to F_(c)-receptors,cells were synchronised at +4° C. for 20 min and the cell-free beadswere removed by low-speed centrifugation (200 g, 10 min, +4° C.). Thecell pellets were resuspended in CM and incubated at 37° C. for 40 minin a CO₂-incubator. After trypsinisation, the cell suspension was takenin sterile conditions, layered over a 7.5% bovine serum albumin (BSA)cushion and centrifuged at 150×g for 10 min at +4° C., to removenon-internalised beads. The cell pellets were resuspended in 0.3 ml of2% PFA in PBS. The results were expressed as the percent of the control,i.e. amount of phagocytic cells containing two or more beads in presencevs. in absence of antibodies, as determined by flow cytometry. AFACScan™ (Becton Dickinson, San Jose, Calif.) with an air-cooled argonlaser providing an excitation at 488 nm was used. A total of 10000events were acquired for each sample and stored in the list mode dataformat. The fluorescence emission was collected at 520 nm (FL1) for thephagocytosis. Data collection and analysis were performed with a Consort30 system and LYSIS-II program. The data were analyzed, once displayedas two-parameter complexity and cell size, in the process of gating andas fluorescence (FL1) frequency distribution histogram to analyze thephagocytosis. EC50, the concentration that induced 50% increase ofphagocytic activity was determined for each antibody tested usingdose-response curves built with the percentages of phagocytic activity,versus the range of concentrations (0.01-5 μg/ml). Then, the EC50 wereextrapolated from these curves and used to compare the relativeefficiency of phagocytic stimulation of the antibodies. The IgGconverted clone 807A-M0028-B02 demonstrated high efficiency (EC50=34±15ng/ml) to stimulate THP-1 cells. The results thus indicate that807A-M0028-B02 specifically directed the in vitro phagocytic uptake ofCTD-bearing beads by human macrophage/microglia-like cells in aconcentration-dependent fashion.

Example 33 Germ Line Correction of Clones Found in Selections Describedin Example 4, Example 21 and Example 22

Of the antibodies described in Table 18, five of them (807A-M0028-B02,807B-M0004-H03, 807B-M0009-F06, 807B-M0004-A03 and 807B-M0079-D10) havebeen investigated further. Somatic mutations in the variable part of thelight chains of these antibodies have been found in all clones. Some ofthe clones also contained mutations in the constant part of the lightchain (Table 21). Sequence alignments with genomic and known germlinesequences have been performed, and the correct amino acids have beenidentified (indicated in bold in Table 19 and Table 20). The VL chainsof the corrected clones are described in Table 19, and the constantparts of the IgG are described in Table 20.

To ensure that the IgG molecules are germline, the somatic mutations arecorrected at the DNA level in the five antibodies, and all fivegermline-corrected IgG1s were expressed (transiently in HEK 293T cells).Comparative binding analysis was performed in Biacore (Example 34),CTD-ELISA and IHC to ensure that the germline-corrected antibodies arestill functional. The results are summarised in Table 23.

Example 34 Biacore Analysis of Germ Line Corrected IgGs

The germline-corrected clones described in Tables 19 and 20 wereanalysed in Biacore. The analysis was performed by running the IgGs atdifferent concentrations over a surface with coated bCTD. A surface witha biotinylated control IgG was used as a negative surface.

Biacore analysis of clones 807A-M0028-B02, 807A-M0028-B02.1 and807A-M0028-B02.2 showed that the three IgG molecules bind to bCTD withsimilar kinetics (similar on-rate and off-rate). The affinity of the IgGis not significantly altered. The same results were obtained whencomparing 807B-M0004-A03 with 807B-M0004-A03.1.

Biacore analysis of 807B-M0004-H03 and 807B-M0004-H03.1 indicated that807B-M0004-H03.1 binds with a different kinetic to the parental clone.However, this did not influence the affinity value significantly. Theclone 807B-M0009-F06.1 had lost its binding capacity to bCTD as shown inCTD-ELISA and Biacore analysis and CTD-ELISA.

Example 35 Binding of Antibodies to ApoE-CTD by ELISA

Antibodies were screened for ApoE-CTD binding capacity using a coatedApoE-CTD ELISA. Human, marmoset or murine ApoE-CTD was coated on amicrotiter plate followed by incubation with test antibodies. Afterthis, the amount of antibody bound was determined by detection withsecondary HRP-antibody and tetramethylbenzidine (TMB) substrate.ApoE-CTD binding gives a high signal measured as optical density (OD) inthe ELISA. Binding of 807A-M0028-B02, 807A-M0028-B02.1 and807A-M0028-B02.2 are exemplified in FIG. 22. Results are disclosed inTable 22.

Example 36 Binding of Antibodies to Human Lipoproteins

Antibodies were screened for lipoprotein binding capacity using a coatedVLDL ELISA. Human VLDL was coated on a microtiter plate followed byincubation with test antibodies. After this, the amount of antibodybound was determined by detection with secondary HRP-antibody andtetramethylbenzidine (TMB) substrate. VLDL binding gives a high signalmeasured as optical density (OD) in the ELISA. Binding of807A-M0028-B02, 807A-M0028-B02.1, 807A-M0028-B02.2, 807B-M0004-H03.0,807B-M0004-H03.1, 807B-M0004-A03 and 807B-M0004-A03.1 are exemplified inFIG. 23. Results are disclosed in Table 22.

Example 37 In Vivo Binding of 807A-M0028-B02, 807B-M0004H03,807B-M0004-A03, 807B-M0079-D110807B-M0009-F06 to Mouse Cerebral Plaquesand In Vitro Binding of Fab Clones to Human AD Cerebral Plaques

In vivo binding of 807A-M0028-B02, B807B-M0004H03, 807B-M0004-A03,807B-M0079-D10 and 807B-M0009-F06 to cerebral plaques (immunodecoration)was demonstrated by i.p. or i.v. injections into APP/PS1 transgenic mice(FIGS. 24 and 25). Immunodecoration was observed already two days aftera single dose administration of 10 mg/kg of 807A-M0028-B02. Binding of807A-M0028-B02, B807B-M0004H03, 807B-M0004-A03, 807B-M0079-D10 and807B-M0009-F06 was only observed on plaques while no staining ofastrocytes or any other brain structure was detected (FIGS. 24 and 25).It was demonstrated that a substantial number of plaques wereimmunodecorated with respective clones by comparing the total plaqueburden in each mouse that was determined by staining with a monoclonalantibody to Aβ (6E10) on adjacent sections.

In vitro binding of affinity maturated clones to amyloid plaques inhuman AD brain sections was also demonstrated by Immunohistochemistry(FIG. 25)

In vitro binding of different wild type clones in human AD plaques isvisualised in FIG. 26.

Example 38 Affinity Maturation of 807A-M0028-B02, 807B-M0004H03,807B-M0004-A03, 807B-M0079-D10807B-M0009-F06 by VH-CDR3 Spiking

Spiking mutagenesis was used to introduce low level mutations over thefall length of the VH-CDR3 of each of 807A-M0028-B02, 807B-M0004H03,807B-M0004-A03, 807B-M0079-D10 and 807B-M0009-F06 in the context of theoriginal wild-type residues (see FIG. 29). PCR was carried out using anoligionucleotide carrying a region of spiked diversity over the lengthof the VH-CDR3 sequence of each antibody bracketed by regions ofhomology with the target V gene in the FR3 and FR4 regions together witha specific primer homologous to the FR1 region capable of annealing tothe 5′ end of the target V gene.

For antibodies 807A-M0028-B02, 807B-M0009-F06 and 807B-M0004-H03,diversification of the VH-CDR3 was realized through a one step PCRamplification. This PCR was performed using a 5′ primer complementary tothe light chain constant region and the 3′specific spikedoligonucleotides. The PCRs were then performed in a volume of 50 μlusing the advantage 2 PCR enzyme system (Clontech) and 10 pmoles of eachprimer for 25 cycles (1 min at 95° C., 1 min at 60° C., and 2 min at 68°C.). A primer concentration of 100 nM was chosen in order to cover theentire diversity carried by the spiked oligonucleotides. 100 to 200reactions were needed to obtain ˜6 μg of PCR products. All products werepurified using the GFX purification kit (Amersham).

The resulting PCR products of 730 bp contain an internal XbaI site andan BstEII site, incorporated in the oligonucleotides. These sites wereused to clone the products into a display vector.

Diversification of the VH CDR3 of 807B-M04-A03 and 807B-M0079-D10 wasrealized in two steps: after primary amplification as described above,the resulting PCR products of 730 bp were re-amplified with acombination of a 5′ end nested forward primer, appended with a SfiIsite, and a 3′end Nhe1-tagged CH1 reverse primer. The PCRs were thenperformed in a volume of 50 μl using the advantage 2 PCR enzyme system(Clontech) and 10 pmoles of each primer for 20 cycles (1 min at 95° C.,3 min at 68° C.); 100 to 200 reactions were needed to obtain ˜6 μg ofPCR products. The number of cycles was kept quite low (20 cycles) inorder to maintain maximal diversity, introduced in the first PCR step.To ensure again maximal diversity, for each reaction, 50 ng of the firstPCR product was used as template to initiate the second PCR reaction.All products were purified using the GFX purification kit (Amersham).

The PCR products and vector backbones were digested using 50 U/μg DNA ofeither XbaI and BstEII (for 807A-M0028-B02, 807B-M0009-F06 and807B-M0004-H03) or SfiI and NheI (for 807B-M0004-A03 and 807B-M79-D10).

The resulting cleaved products (both vectors and PCR fragments) weregelpurified, 1.6 μg of each DNA fragment was ligated into 10 μg ofsimilarly cut phagemid vector backbone using T₄ DNA ligase (NEB) and theligation mixture for each spiked library was introduced into E. coli TG1cells by electroporation.

Phagemid particles were rescued from the libraries using helper phageM13-K07 (Marks et al., (J. Mol. Biol. 222, 581 (1991)) using enoughbacteria from each library for inoculation in order to represent eachclone at least once.

The diversity in the VH-CDR3 of each library was evaluated bysequencing. 96 isolates were randomly picked for each library and theVH-CDR3 regions were sequenced and compared to the reference wild typeVH-CDR3 sequences.

For clones of 807B-M0079-D10 and 807B-M0004-A03 the full VH sequence wasdetermined and compared to the VH reference regions outside VH-CDR3.

A selection procedure consisting of two rounds of selection was used topreferentially enrich the higher affinity clones in the library over thelower affinity clones. The first round of affinity selection was carriedout using a reduced antigen concentration relative to the concentrationused to select the wild-type antibody. The optimum reduced antibodyconcentration was determined empirically using the wild-type antibodyand a control antibody. The second round was performed at a furtherreduced antigen concentration in the presence of competing soluble Fabor IgG. The selection conditions used are detailed in Table 3.

To determine if enrichment of antigen positive clones had occurred, 46randomly picked clones from before and after the first round ofselection for each antibody to be matured were tested in an antigenELISA. In all cases, enrichment of antigen positive clones after justone round of selection was observed.

After selection, the geneIII stump was removed from the vector to allowsoluble Fab expression. 200 clones were randomly picked, screened byELISA and their heavy chains sequenced. The Dyax WEBPHAGE database wasused to link ELISA data to the respective sequences. The VH-CDR3sequences of the clones found to be positive in the screening assay (ODsignal=3× background) were analysed further.

For the clones 807B-M0079-D10 and 807B-M0004-A3 the full VH region wasamplified and cloned and so the full VH sequence was obtained andcompared to the VH reference sequence in order to check for anymutations outside VH CDR3. Clones containing mutations in frameworkregions were discarded but clones with mutations in the VH-CDR1-CDR2were kept.

The results of amino acid frequency analysis of selected clones arepresented in Tables 24 to 30. In the VH-CDR3, some amino acid positionsare very conserved whilst others are frequently mutated.

Biacore screening was used to select five CDR3-mutated Fabs based on offrate or K_(D). CDR3-mutated Fabs were expressed in bacteria Periplasmicextracts were prepared and screened in Biacore. The best clones wereselected based on either off-rates or K_(D) for binding to hCTD or apeptide as shown in Table 4.

The biotinylated peptides or human CTD were coated on streptavidinchips. Periplasmic extracts from 10 ml cultures were diluted ½ inHBS+0.1% BSA. Samples were injected at 30 μl/min for 3 minutes using thekinject program. Following a 3 minute dissociation, any remaining samplewas stripped from chip surface. Off rates were measured on a time windowof 1 min (between 10-70s). These data are presented in Tables 31 to 35.

On rates can be calculated from Biacore curves if both k_(off) and theFab concentration are known. Under conditions of full Mass TransferLimitation (MLT) like those encountered when working with very highdensity chips and low flow rates, the Biacore signal depends only on theconcentration of the analyte run over the chip surface. Fabconcentrations in crude samples can be determined from a standard curveobtained by running different concentrations of a purified Fab on a highdensity Protein A chip. Using the Fab concentrations obtained that wayand the k_(off) values, we calculated k_(on) data from the Biacorecurves. The equilibrium dissociation constant K_(D) was obtained fromk_(off)/k_(on) These data are presented in Tables 31 to 35. Note thatthe 807B-M0079-D10 does not bind to Protein A. Therefore, only k_(off)values are presented for this clone.

Five variants were successfully selected for 807B-M0004-A03,807B-M0004-H03, 807B-M0009-F06 and 807A-M0028-B02. The selected variantsare identified in Table 36. No clones with significantly improvedK_(off) were found for 807B-M0079-D10 (no K_(D) data available for thisclone).

The selected Fabs were produced in E. coli and purified from periplasmicextracts by Immbolized Metal Affinity Chromatography. The quality of thepreparations was checked on reducing and non reducing SDS-PAGE.

The purified Fabs were used to accurately determine the equilibriumdissociation constant K_(D).

Biotinylated peptides or human CTD were coated on streptavidin chips.Experiments were performed in HBS running buffer. Purified Fabs werediluted to 200 nM and serial ½ dilutions to 12.5 nM were made and run induplicates. For association, samples were injected at 30-40 μl/min usingthe kinject program. Following a 3 minute dissociation, any remainingsample was stripped from the chip surface. Sensorgrams were analyzedusing the simultaneous ka/kd fitting program in the BIAevaluationsoftware 3.1. The data are summarized in Table 36. The best clonesselected from Biacore screening exhibit an affinity 2 to 3-fold higherthan the original clone for 807B-M0004A03, 807B-M0009-F06 and807A-M0028-B02. In the case of 807B-M0004-H03, the Biacore signals didnot allow an accurate comparison of the different clones.

Purified Fabs were also tested by immunohistochemistry. Table 37 showsthe names of the chosen clones together with the wild type clones andindicates whether they stain plaques in immunohistochemistry.

Example 39 Light Chain Shuffling of the Antibody Variants Selected fromExample 38

As a starting point for light chain shuffling (cycle 2 in FIG. 29), theheavy chains corresponding to the VH-CDR3 improved variants from Example38 (cycle 1 in FIG. 29) were used together with wild-type (WT) clones.

The WT clone 807B-M0009-F06 was not included because the affinity ofthis clone was significantly lower compared to the selected variantsfrom cycle 1. For antibody 807B-M0079-D10, as no improved affinityvariant was found in cycle 1, the LC shuffling was performed on the WTclone alone.

In this Example, the non-affinity matured antibodies have beendesignated as the wild type (WT) clone and the selected variants fromcycle 1 as parental clones.

The selected heavy chain variants from cycle 1 were cloned into theFAB310 vector backbone containing a repertoire of 5 to 6 heaving chains(HC) shuffled with approximately 10⁸ light chains (LC) to createcombinatorial diversity.

For every clone, a Qiagen DNA preparation was performed on a TG1culture. 10 μg of DNA was then cleaved using SfiI and NotI restrictionenzymes, generating a heavy chain fragment size of 650 bp. The FAB310vector backbone was similarly cut.

The resulting cleaved products (both vector and fragments) weregelpurified and, for each library, the different heavy chain variantfragments were pooled in equal amounts and 3 μg of the pooled fragmentswere ligated with 6 μg of cleaved phagemid vector backbone using T₄ DNAligase (NEB). Desalted ligation mixtures for each library wereintroduced into E. coli TG1 cells by electroporation.

The library sizes achieved were such that each heavy chain variant wascombined with at least one copy of each member of the light chainrepertoire.

Heavy chain sequences were determined for 50 isolates, randomly pickedfrom each library.

The light chain sequences of 48 isolates randomly picked from the807A-M0028-B02-derived unselected library and 48 from the 807B-M0009-F06derived unselected library. 63 unique functional light chains wereobtained.

Phagemid particles were rescued with helper phage M13-KO7 (Marks et al.J. Mol. Biol. 222, 581 (1991)) using enough bacteria from each libraryfor inoculation in order to represent each clone at least once.

The 5 light chained shuffled libraries were selected for improvedaffinity variants. Prior to selections the libraries were depleted forstreptavidin binding antibodies by pre-incubating the libraries with 100μl streptavidin paramagnetic beads in 1 ml 2% MPBS. For each librarythree concentrations of antigen were used to determine the optimalconcentration for the second round of selection (Table 5). Theincubation time of the phage with bead-target complex was reduced to 0.5hour and 11 cycles of programmed washing was used in the Kingfisherdevice. After selection the bound phage were eluted and infected with E.coli (TG1 OD of 0.5) and the liquid amplified overnight at 30° C. withshaking at 250 rpm in 25 ml 2×TY/Ampiclillin (100 ug/ml) Glucose (2%w/v). Cells were concentrated and glycerol stocks were made in order toperform the round 2 selection.

Unselected library and output was titrated to get single colonies forpicking and screening. From the unselected library and output library 47colonies were picked and screened in a phage ELISA (Coated antigen 0.5μg/ml for all antigens via b-BSA plus streptavidin).

All selection arms resulted in the enrichment of antigen binding clones.Based on these results an antigen concentration was chosen for thesecond round of selection.

The conditions for the round 2 selection were chosen to be morestringent and were designed to select for improved (faster) k_(on) andimproved (slower) k_(off). Three strategies were used as outlined inTable 6: Strategy I—further lowering of antigen concentrations; strategyII—further lowering of antigen concentration and reduced incubation timewith antigen (k_(off) selection); strategy III—further lowering ofantigen concentration and increased stringency washing (k_(off)selection). Selection was performed in a KingFisher automated device andan input of approximately 10¹² phages were used. Prior to selections thelibraries were depleted for streptavidin binding antibodies as describedabove. After selection the bound phage were eluted and used to infect E.coli as described previously. Cells were concentrated and glycerolstocks were made.

The output of round 2 was pre-screened in ELISA to determine thepercentage of antigen binding clones. Sequence analysis of a limitednumber of clones was performed to determine if any particular clone wasdominating selection and if there are any dominant light chain familiesin the selected clones.

Each of the round 2 phage outputs (15 in total) were re-cloned in batchin order to produce soluble Fabs. This was achieved through the removalof the geneIII stump from the vector.

200 clones were randomly picked and screened by ELISA for their bindingto their respective antigen. Antibody sequences were determined for thepositive hits only. The storage and initial sequence analysis wasconducted via Dyax WEBPHAGE database.

For libraries 807B-M0004-A03, 807B-M079-D10 and 807A-M0004-H03,antibodies enriched between 2 to 8 times were selected. For libraries807A-M0028-B02 and 807B-M009-F06, all clones were taken since number ofunique clones was low. The light and heavy chain sequences from allELISA positive and unique hits are shown in Tables 38 to 42.

After Biacore analysis of the potential affinity matured binders, a fewwere found to have a higher affinity. Only libraries 807B-M0004-A03,807A-M0028-B02 and 807B-M004-H03 gave affinity matured antibodies with ahigher affinity than the WT and parental clones. The light chainsequence of those selected clones was aligned to the germline.Interestingly, the same amino acid positions seem to be diversifiedamong all the selected clones belonging to the same germline. The sameamino acid positions seem to be diversified among all the selectedclones belonging to the same germline.

For library 807B-M0004-A03, although clones M148E08 and M150E03 haveexactly the same heavy chain sequence and light chains which differ bytwo amino acids, clone M150E03 has a 3.2× improved affinity compared toM148E08, suggesting that only two amino acids located in FR1 areresponsible for this improvement.

For libraries 807B-M0028-B02 and 807B-M0004-A03, most of thediversification observed in the CDR and FR was reversion to the germlinesequence compared to wild-type.

Only a few conservative variations were observed in FR3 of 807A-M0028B02 library-derived clones.

For Biacore analysis, all clone variants were grown in small cultures(typically 10 ml), periplasmic extracts (PE) were prepared and the Fabconcentration in the PEs was measured by running the samples on aProtein A/G chip. The PEs were then diluted to the same Fabconcentration (25-50 nM) and run over a target-coated chip (peptide 4,8, 9 or CTD). The best clones were identified based either on theamplitude of the association and dissociation phases (807A-M0028-B02,807B-M0004-A03, 807B-M0009-F06) or on the value of the Biacore signal atequilibrium (807B-M0004-H03). Clones derived from 807B-M0079-D10 do notbind to Protein A/G chips and were thus ranked only based on theiroff-rates

Clones derived from 807B-M0004-H03 were ranked based on the value of theBiacore signal at equilibrium which reflects the equilibriumdissociation constant K_(D): the higher the Biacore signal atequilibrium, the better the affinity.

Clones derived from 807B-M0079-D10 were ranked based only on k_(off).

Four and five variants were selected for 807A-M0028-B02 and807B-M0004-A03, respectively. None of the isolated 807B-M0009-F06variants seemed to exhibit an affinity higher than the best variantsobtained from CDR3 spiking. For 807B-M0004-H03, two variants wereselected based on the value of the Biacore signal at equilibrium. Noneof the 807B-M0079-D10 variants could be selected based on off-rateanalysis.

The selected clones selected were produced in E. coli and purified fromperiplasmic extracts. This material was used to measure accurately theequilibrium dissociation constant K_(D) in Biacore. The data aresummarized in Table 7. The best clones isolated from light chainshuffling exhibit an affinity ˜5-times better than the correspondingoriginal wild type clones or the best clones isolated from CDR3 spiking.

The clones selected following light chain shuffling are shown in Tables43 and 44.

TABLE 1 Off-rate measurement of sFabs originating from selections onfibrils and ur-bCTD koff Initial name HCDR3 (e⁻³s⁻¹) RU 807A-M0027-C11AVGYGDYGDY 13.30 79.2 807A-M0027-H05 DFFTSYFDH 16.90 182.0807A-M0026-F11 DLWFGEWDY 28.10 165.8 807A-M0026-H09 DLWFGEWDY 25.10138.0 807A-M0027-E12 DLWFGEWDY 8.95 419.9 807A-M0028-B12 DLWFGEWDY 18.80335.5 807A-M0029-G10 DLWFGEWDY 23.80 212.0 807A-M0027-G01 DRGVSLLGAFDI30.00 231.3 807A-M0028-A07 ESIAVAGVDY 53.60 367.0 807A-M0026-F05GRGNYDFWSAGYYYYYMDV > 158.0 807A-M0028-G07 QEVWQWPAQFDS 35.30 131.7807A-M0027-E11 SLDLDY 40.50 412.3 807A-M0026-G08 SSGIYYGYYMDV 38.80594.1 807A-M0028-B02 SVLLDY 28.00 454.6 807A-M0028-B06 DRGVSLLGAFDI13.50 100.3 807A-M0027-D05 EPIWGYYYYGMDV 9.16 377.8

TABLE 2 Comparison of Fab and IgG binding on CTD-coated chip for thecandidate clones 807A-M0026-F05 (26F5), 807A-M0027-E11 (27E11) and807A-M0028-B02 (28B2) ka (1/Ms) kd (1/s) Clone Format Surface e5 e−3 KD(nM) 27E11 Fab HD 9.5 45.5 47.8 ± 8.1  IgG HD 7.5 7.4* 9.9 ± 4.1 8.12.1^(#) 2.6 ± 1.1 LD 10.1 7.7* 7.6 ± 3.2 11.1 2.1^(#) 1.9 ± 0.8 28B2 FabHD 2.5 44.2 179 ± 5.7  IgG HD 3.7 4.0 10.8 ± 2.4  LD 5.2 6.4 12.3 ± 3.7 26F5 Fab HD n.f. n.f. n.d. IgG HD 0.2 42 2103 ± 785  LD n.f. n.f. n.d.*kd measured directly after injection stop. ^(#)kd measured ~50 secafter injection stop. n.f. no fit n.d. not determined

TABLE 3 Antibody Round 1 selection Round 2 selection 807A-M0028-B02 3 nMbCTD or 0.3 nM bCTD + 5 μM wt Fab 1 tube of fibrils or 0.3 nM IgG1807B-M0004-A03 5.7 nM p4 0.057 nM p4 + 5 μM wt Fab or 0.01 nM IgG1807B-M0004-H03 5.7 nM p4 0.57 nM p4 + 5 μM wt Fab or 0.3 nM IgG1807B-M0009-F06 5.7 nM p9 0.057 nM p9 + 5 μM wt Fab or 0.3 nM IgG1807B-M0079-D10 5.7 nM p8 0.057 nM p8 + 5 μM wt Fab or 0.3 nM IgG1

TABLE 4 Number of clones Original clone K_(D) (nM) screened807A-M0028-B02 150 (on hCDT) 72 807B-M0004-A03  98 (on p4) 39807B-M0004-H03 200 (on p4) 54 807B-M0009-F06 172 (on p9) 24807B-M0079-D10  26 (on p8) 33

TABLE 5 Percentage of antigen binding clones retrieved from the round 1selections at decreasing antigen concentrations Library Round Antigen Agconcentrations A03 1 P4 57 nM, 5.7 nM, 0.57 nM Hit rate % Input 2% 2% 2%Output 62%  51%  83%  H03 1 P4 570 nM,  57 nM, 5.7 nM Hit rate % Input2% 0% 0% Output 72%  72%  81%  F06 1 P9 57 nM, 5.7 nM, 0.57 nM Hit rate% Input 0% 0% 0% Output 6% 4% 2% D10 1 P8 57 nM, 5.7 nM, 0.57 nM Hitrate % Input 0% 0% 0% Output 34%  45%  38%  B2 1 CTD 300 nM,   30 nM,  3 nM Hit rate % Input 2% 2% 2% Output 28%  21%  17% 

TABLE 6 Conditions used for the second round selection strategies Agconc Incubation Strategy Library Round Antigen (nM) time (mins) WashingI A3 2 Pep-4 0.057 30 11 × 5 mins I H3 2 Pep-4 5.7 30 11 × 5 mins I D102 Pep-8 0.057 30 11 × 5 mins I B2 2 b-CTD 10 30 11 × 5 mins I F6 2 Pep-95.7 30 11 × 5 mins II A3 2 Pep-4 0.57 3 11 × 5 mins II H3 2 Pep-4 5.7 311 × 5 mins II D10 2 Pep-8 0.57 3 11 × 5 mins II B2 2 b-CTD 30 3 11 × 5mins II F6 2 Pep-9 5.7 3 11 × 5 mins III A3 2 Pep-4 0.57 30 3 × 5 mins +3 × 45 mins + 3 × 5 mins III H3 2 Pep-4 5.7 30 3 × 5 mins + 3 × 45mins + 3 × 5 mins III D10 2 Pep-8 0.57 30 3 × 5 mins + 3 × 45 mins + 3 ×5 mins III B2 2 b-CTD 30 30 3 × 5 mins + 3 × 45 mins + 3 × 5 mins III F62 Pep-9 5.7 30 3 × 5 mins + 3 × 45 mins + 3 × 5 mins

TABLE 7 Clone name k_(on) (1/Ms) k_(off) (1/s) K_(D) (nM) M0004A03 WT4.19E+05 3.31E−02 79 * M0004A03-M0148-E08 9.51E+05 4.85E−02 51 *M0004A03-M0149-F02 6.51E+05 2.41E−02 37 * M0004A03-M0149-G11 4.12E+051.73E−02 42 * M0004A03-M0150-E03 6.08E+05 9.89E−03 16 *M0004A03-M0151-D09 5.35E+05 1.61E−02 30 * M0028B02-M0168-D10 6.97E+042.97E−03 43 * M0028B02-M0169-F03 1.73E+05 1.78E−03 10 *M0028B02-M0171-E03 1.02E+05 2.61E−03 26 * M0028B02-M0171-G02 8.99E+044.55E−03 51 * * Kinetic analysis, 1:1 model

TABLE 8 Description of SEQ ID NOS: 21-164 and 171-206 Antibody NameVH-CDR1 VH-CDR2 VH-CDR3 VH VL-CDR1 807A-M0027-E11 SEQ ID NO: 21 SEQ IDNO: 22 SEQ ID NO: 23 SEQ ID NO: 39 SEQ ID NO: 30 807A-M0028-B02 SEQ IDNO: 24 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 40 SEQ ID NO: 33807A-M0026-F05 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 41SEQ ID NO: 36 807B-M0001-B07 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47SEQ ID NO: 135 SEQ ID NO: 90 807B-M0004-A03 SEQ ID NO: 48 SEQ ID NO: 49SEQ ID NO: 50 SEQ ID NO: 136 SEQ ID NO: 93 807B-M0004-A05 SEQ ID NO: 51SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 137 SEQ ID NO: 96 807B-M0004-C04SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID NO: 56 SEQ ID NO: 138 SEQ ID NO: 99807B-M0004-C05 SEQ ID NO: 57 SEQ ID NO: 58 SEQ ID NO: 59 SEQ ID NO: 139SEQ ID NO: 102 807B-M0004-F06 SEQ ID NO: 60 SEQ ID NO: 61 SEQ ID NO: 62SEQ ID NO: 140 SEQ ID NO: 105 807B-M0004-F10 SEQ ID NO: 63 SEQ ID NO: 64SEQ ID NO: 65 SEQ ID NO: 141 SEQ ID NO: 108 807B-M0004-H03 SEQ ID NO: 66SEQ ID NO: 67 SEQ ID NO: 68 SEQ ID NO: 142 SEQ ID NO: 111 807B-M0009-C03SEQ ID NO: 69 SEQ ID NO: 70 SEQ ID NO: 71 SEQ ID NO: 143 SEQ ID NO: 114807B-M0009-F06 SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 74 SEQ ID NO: 144SEQ ID NO: 117 807B-M0013-A12 SEQ ID NO: 75 SEQ ID NO: 76 SEQ ID NO: 77SEQ ID NO: 145 SEQ ID NO: 120 807B-M0079-D10 SEQ ID NO: 78 SEQ ID NO: 79SEQ ID NO: 80 SEQ ID NO: 146 SEQ ID NO: 123 807B-M0081-F12 SEQ ID NO: 81SEQ ID NO: 82 SEQ ID NO: 83 SEQ ID NO: 147 SEQ ID NO: 126 807B-M0081-H03SEQ ID NO: 84 SEQ ID NO: 85 SEQ ID NO: 86 SEQ ID NO: 148 SEQ ID NO: 129807B-M0083-E11 SEQ ID NO: 87 SEQ ID NO: 88 SEQ ID NO: 89 SEQ ID NO: 149SEQ ID NO: 132 Antibody Polynucleotide Polynucleotide Name VL-CDR2VL-CDR3 VL encoding VH encoding VL 807A-M0027-E11 SEQ ID NO: 31 SEQ IDNO: 32 SEQ ID NO: 42 SEQ ID NO: 174 SEQ ID NO: 173 807A-M0028-B02 SEQ IDNO: 34 SEQ ID NO: 35 SEQ ID NO: 43 SEQ ID NO: 176 SEQ ID NO: 175807A-M0026-F05 SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 44 SEQ ID NO: 172SEQ ID NO: 171 807B-M0001-B07 SEQ ID NO: 91 SEQ ID NO: 92 SEQ ID NO: 150SEQ ID NO: 178 SEQ ID NO: 177 807B-M0004-A03 SEQ ID NO: 94 SEQ ID NO: 95SEQ ID NO: 151 SEQ ID NO: 180 SEQ ID NO: 179 807B-M0004-A05 SEQ ID NO:97 SEQ ID NO: 98 SEQ ID NO: 152 SEQ ID NO: 182 SEQ ID NO: 181807B-M0004-C04 SEQ ID NO: 100 SEQ ID NO: 101 SEQ ID NO: 153 SEQ ID NO:184 SEQ ID NO: 183 807B-M0004-C05 SEQ ID NO: 103 SEQ ID NO: 104 SEQ IDNO: 154 SEQ ID NO: 186 SEQ ID NO: 185 807B-M0004-F06 SEQ ID NO: 106 SEQID NO: 107 SEQ ID NO: 155 SEQ ID NO: 188 SEQ ID NO: 187 807B-M0004-F10SEQ ID NO: 109 SEQ ID NO: 110 SEQ ID NO: 156 SEQ ID NO: 190 SEQ ID NO:189 807B-M0004-H03 SEQ ID NO: 112 SEQ ID NO: 113 SEQ ID NO: 157 SEQ IDNO: 192 SEQ ID NO: 191 807B-M0009-C03 SEQ ID NO: 115 SEQ ID NO: 116 SEQID NO: 158 SEQ ID NO: 194 SEQ ID NO: 193 807B-M0009-F06 SEQ ID NO: 118SEQ ID NO: 119 SEQ ID NO: 159 SEQ ID NO: 196 SEQ ID NO: 195807B-M0013-A12 SEQ ID NO: 121 SEQ ID NO: 122 SEQ ID NO: 160 SEQ ID NO:198 SEQ ID NO: 197 807B-M0079-D10 SEQ ID NO: 124 SEQ ID NO: 125 SEQ IDNO: 161 SEQ ID NO: 200 SEQ ID NO: 199 807B-M0081-F12 SEQ ID NO: 127 SEQID NO: 128 SEQ ID NO: 162 SEQ ID NO: 206 SEQ ID NO: 205 807B-M0081-H03SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 163 SEQ ID NO: 202 SEQ ID NO:201 807B-M0083-E11 SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 164 SEQ IDNO: 204 SEQ ID NO: 203

TABLE 9 Amino acid sequences of the VL chains of the antibodiesidentified using the screening strategy of Example 5 Initial Name LV-FR1LV-CDR1 LV-FR2 LV-CDR2 807A-M0027-C11 QDIQMTQSPSTLSASVGD RASQSVSSWLAWYQQKPGAAPRLLIY KASSLQT RVTITC 807A-M0043-F08 QDIQMTQSPSSVSASVGDRASQGISSWLA WYQQKPGKAPKLLIY AASSLQS RVTITC 807A-M0039-E07QSELTQPPSASGSPGQSV TGTSSDVGGYNYVS WYQQHPGKAPKLMIY EVSKRPS TISC807A-M0039-D11 QDIQMTQSPSSLSASVGD QASQDIRNYIN WYQQKPGKAPKLLIN DASNLEPRVTMTC 807A-M0037-F10 QSALTQPPSASGTPGQRV SGRSSNIGSNSVN WYQQLPGTAPKLLIYSNNQRPS TISC 807A-M0028-B06 QDIQMTQSPDTLSLSPGDR RASQSVSSNYLAWYHQKPGQAPRLVIY NTSRRAT ATLSC 807A-M0046-F05 QDIQMTQSPGTLSLSPGERRASQSVSSSYLA WYQQKPGQTPRLLIY GASSRAT ATLSC 807A-M0041-F03QDIQMTQSPSTLSASVGD RASQGINRWLA WYQQKPGKAPKLLIY KASALES RVAITC807A-M0043-E08 QYELTQPPSASGSPGQSV TGTSSDVGAYNYVS WYQQHPGKAPKLIIY EVNKRPSTISC 807A-M0042-D05 QDIQMTQSPATLSVSPGD RASQSVGSTLA WYQQKPGQAPRLLIYGAVTRAT RVTLSC 807A-M0029-G10 QDIQMTQSPSSLSASVGD RASQSISSYLNWYQQKPGKAPKLLIY AASSLQS RVTITC 807A-M0046-G03 QDIQMTQSPSSLSASVGDRASQGITNWVA WYQQKPGKAPKLLIY GASRLQS RVTITC 807A-M0037-D06QDIQMTQSPSSLSASVGD RTSQDVRNWVA WYQQKPGKAPNLLIY MASTLQS RVTITC807A-M0043-E07 QDIQMTQSPSSLSASVGD RASQNVNTFLN WYQHKAGKAPKLLIY AASSLQSRVTITC 807A-M0027-E11 QDIQMTQSPSSLSASVGD RASQRIRKNLH WYQQKPGKAPNLLIYDASSNER RVTITC 807A-M0046-A11 QDIQMTQSPSSLSASVGD RASQSISSYLNWYQQKPGKAPKLLIY AASSLQS RVTITC 807A-M0041-E01 QDIQMTQSPSSLSASVGDHASQDIANYLS WYQQKPGKAPKLLIY DAFNLET RVTITC 807A-M0044-B07QDIQMTQSPGILSLSPGER RASQNLIFNFLA WYQHKPGQAPRLLIY GSSTRAT ATLSC807A-M0028-B02 QDIQMTQSPSSLSASVGD RTSQDIRNHLG WFQQKPGKAPQRLIR EASILQSRVTITC 807A-M0039-E06 QDIQMTQSPSSVSASVGD RASQGISSWLA WYQQKPGKAPKLLIYAASSLQS RVTITC 807A-M0040-A03 QSELTQPPSVSVSPGQTA SGDKLGDKYASWYQQRPGQSPVLVIY QDTKRPS TITC 807A-M0044-G07 QDIQMTQSPSSVSASVGDRASQVISTWLS WYQQKPGKAPKLLIY TASTLQS RVTITC 807A-M0044-E08QSPSSVSTSVGDRVTITCR LDIQMTDISTWLA WYQQKPGKAPKLLIY AASTLES ASQ807A-M0038-A09 QSALTQPPSVSVAPGQTA GGNNIGTKIVN WYQQRPGQAPVVVVY DNSDRPSRITC 807A-M0037-C08 QDIQMTQSPSSVSASVGD RASQGISSWLA WYQQKPGKAPKLLIYAASSLQS RVTITC 807A-M0039-H09 QDIQMTQSPSSLPASVGD RTSQSISDYVNWYQQKPGKAPNLLIY AASTLQG SVTVTC 807A-M0039-D05 QDIQMTQSPSSLSASVGDRASQDIRDDLG WYQQKPGKAPKRLIY AASSLQS RVTITC 807A-M0042-F12QSELTQPPSASGTPGQRV SGGYSNMGSNYAH WYQQLPGTAPKLLIY NNNQRPS TISC807A-M0043-H05 QSELTQPASVSGSPGQSIT TGTNTDVGGYNYVA WYQQHPGKAPKLMIYDVSNRPS ISC 807A-M0042-C03 QDIQMTQSPATLSLSPGER RASQSVSSSYLAWYQQKPGQAPRLLIY GASSRAT ATLSC 807A-M0040-C03 QDIQMTQSPGTLSLSPGERRASQIFSSSYVA WYQQKPGQAPRLLIY GASSRAS ATLSC 807A-M0046-C05QDIQMTQSPSSLSASVGD RASQSISSYLN WYQQKPGKAPKVLIY GTSSLQS RVTITC807A-M0027-D05 QDIQMTQSPSSLSASVGD RASQSISSYLN WYQQKPGKAPKLLIY AASSLQSRVTITC 807A-M0040-B11 QSVLTQPPSASGTPGQRV SGSSSNIGSNNVN WYQQLPGTAPKLLIYSNDQRPS TISC 807A-M0039-B02 QDIQMTQSPSTLSASVGD RASQSISSWLAWYQQKPGKAPKLLIY TASSLES RVTITC 807A-M0041-C07 QDIQMTQSPSSLSASLGDRRASQGISNSLA WYQQKPGKAPKLLIS AASTLQT VTITC 807A-M0041-H04QSELTQPPSASGTPGQRVI SGSGSNIGSNIVS WFQQLPGAAPRLLIY NDHRRPS ISC807A-M0028-G07 QYELTQPPSVSVAPGQTA GGNNIGSKNVH WYQQKPGQAPVLVVY DDTDRPSRITC 807A-M0041-A09 QDIQMTQSPATLSLSPGER RASQSVSNNLA WYQQKPGQAPRLLISGASTRAT ATLSC 807A-M0042-B10 QSALTQPASVSGSPGQSIT SGTDSDVGGYNHVSWYQQHPGKAPKLIIY DVDHRPS ISC 807A-M0041-E06 QSVLTQPPSTSGTPGQRVSGSNSNIGSKTVN WYQQLPGTAPKLLIY MNYERPS TISC 807A-M0037-D10QDIQMTQSPSSLSASVGD RASQSIYTSLN WYQQKPGKAPRLLIS DASNLQS RVTITC807A-M0044-F04 QDIQMTQSPATLSVSPGG RASQSVRKNVA WYQQKPGQPPRLLIY GASTRATRATLSC 807A-M0043-D10 QDIQMTQSPATLSVSPGE RASQSVSSGLA WYQQKPGQSPRLLIYDISTRAT GATLSC 807A-M0043-G06 QDIQMTQSPSYLSASVGD RASQTISRYLNWYQQKPGNAPKLLIY AASTLQS RVTITC 807A-M0037-G01 QDIQMTQSPATLSVSPGERRASQSVSSNLA WYQQKPGQAPRLLIY GASTRAT ATLSC 807A-M0044-E11QSALTQPASVSGSPGQSIT TGTSTDVGGYNYVS WYQKHPGKAPKLMIY DVSNRPS ISC807A-M0043-A10 QDIQMTQSPSSLSASVGD RASQSISSYLN WYQQKPGKAPKLLIY TTSFVQSRVTITC 807A-M0045-B03 QYELTQPASVSGSPGQSIT TGTSSDVGAFNYVS WYQHHPGKAPKLLLYEVTNRPS ISC 807A-M0038-A08 QDIQMTQSPSSLSASVGD RASQSIRIYLNWYQQKPGKAPKLLIY AASKLED RVTITC 807A-M0039-C02 QDIQMTQSPGTLSLSPGDRASQSVGSDYLA WYQQKPGQAPRLLIF AASTRAT RATLSC 807A-M0027-G01QSELTQPPSASGTPGQRV SGGYSNMGSNYAH WYQQLPGTAPKLLIY NNNQRPS TISC807A-M0039-B08 QDIQMTQSPSSLSASVGD RASQGISNFLA WYQQKPGKAPKVLIY DASTLRSRVTITC 807A-M0048-E12 QDIQMTQSPSSLSASVGD RASQGIRNDLG WYQQKPGKAPKRLIYGASSLQS RVTITC 807A-M0041-A08 QSELTQPASVSGSPGQSIT TGTSSDVGGYNYVSWYQQHPGKAPKLMIY DVSNRPS ISC 807A-M0037-C09 QDIQMTQSPSSLSASVGDRASQGIRNNLA WYQQRPGKAPKRLIY GASNLHS RVTVTC 807A-M0040-G01QDIQMTQSPSSLSASVGD RASQGIHNYVN WYQQKPGKAPKLLIY AASSLQS RVTITC807A-M0045-E04 QDIQMTQSPSSLSASVGD RASQGIRKDLG WYQQRPGKAPKLLIY GASSLLNRVTITC 807A-M0041-H05 QSALTQPPSVSGAPGQRV TGSSSNIGAPYDVH WYQQVPGTAPKVLIYGNNHRPS TISC 807A-M0043-A08 QSALTQPASVSGSPGQSIT TGTSNDVGGYNSVSWYQQHPGKAPKLLIY DVTNRPS ISC 807A-M0038-C09 QDIQMTQSPDSLAVSLGERKSSQSVLYSSNNKN WYQQKPGQPPKLLIY WASIRES ATINC YLA 807A-M0042-F09QDIQMTQSPSSLSASVGD RASQGIRHDLG WYQQKPGKAPKRLIY AASSLQN RVTITC807A-M0045-B12 QDIQMTQSPSSLSASVGD RTSQNINTYLN WYQQKPGKAPRLLIY AASSLQSRVTITC 807A-M0044-C04 QDIQMTQSPSSLSASVGD RASQTISNYLN WYQQKPGKAPKLLIYATSTLQS RVTITC 807A-M0026-F11 QSVLTQPASVSGSPGQSIT TGTSSDVGIYNYVSWYQQHPGKAPKLMIY DVSNRPS ISC 807A-M0027-E12 QDIQMTQSPGTLSLSPGERRASRSLFSTYLA WYQQKPGQPPRLLIY GASTRAT ATLSC 807A-M0028-B12QDIQMTQSPLSLSASAGDR RASQNINRYLN WYQQKPGKAPRLLIY AASNLQS VTITC807A-M0026-G08 QDIQMTQSPSSLSASAGD RANQGIRNNLA WFQQKPGKAPKSLIY DASSLQSRVTITC 807A-M0043-A07 QDIQMTQSPSSVSASVGD RASQGISSWLA WYQQKPGKAPKLLIYAASSLQS RVTITC 807A-M0042-F04 QDIQMTQSPATLSLSPGES RASQSVNDYLAWYQQKPGQAPRLLIY DSSNRAT ATLSC 807A-M0045-H09 QDIQMTQSPSSLSASVGDRASQSISSYLN WYQQKPGKAPKLLIY VASSLQS RVTITC 807A-M0046-D04QDIQMTQSPGTLSLSPGES RASQSISSYLN WYQQKQGKAPKLLMF AASSLKS ATLSC807A-M0040-G04 QDIQMTQSPATLSASVGD RASQSISSYLN WYQQKPGKAPKLLIY AASSLQSRVTITC 807A-M0045-B01 QSVLTQPASVSGSPGQSIT TGTNTDVGGYNYVS WYQQNPGEAPKLIIYEVNHRPS ISC 807A-M0040-A08 QSVLTQPPSASGTPGQRVI SGSSSNIGSNIVSWFQQVPGAAPRLLIY NDHRRPS ISC 807A-M0026-F05 QDIQMTQSPGTLSLSPGERRASQSIGSRYLA WYQQKPGQAPRLLIY DASKRAT ATLSC 807A-M0037-H02QDIQMTQSPGTLSLSPGER RASQSVSSSYLA WYQQKPGQAPSLLIY DMSTRAP ATLSC807A-M0042-A06 QSALTQPPSASGTPGQRV SGGYSNMGSNYAH WYQQLPGTAPKLLIY NNNQRPSTISC 807A-M0028-A07 QDIQMTQSPSSLSASIGDR RASQGISNYLA WYQQKPGKVPNLLIYAASTLQS VTITC 807A-M0043-G01 QDIQMTQSPSTLSASVGD RPSQSTSNWLAWYQQKPGKAPKLLIY KASILES RVTITC 807A-M0046-F04 QDIQMTQSPSSLSASVGDRASQSISTYLN WYQHKPGNAPNLLIY GASSLKR RVTITC 807A-M0037-F03QDIQMTQSPGTLSLSPGER RASQSISSRYLA WYQQKAGQAPRLLMY GASRAT ATLSC807A-M0039-C10 QSALTQPRSVSGSPGQSV TGTYSDVGNYYSVS WYQQHPGKAPKFIIY DVTKRPSTISC Initial Name LV-FR3 LV-CDR3 LV-FR4 807A-M0027-C11GVPSRFSGGGSGTEFTLTISSLQP QQSYSTPT FGGGTKVEIK DDFATYYC 807A-M0043-F08GVPSRFSGSGSGTDFTLTISSLQP QQANSFPPT FGQGTKVEIQ EDFATYYC 807A-M0039-E07GVPDRFSGSKSVTSASLAITGLQA QSYDSSLSGYV FGSGTKVTVL EDEADYYC 807A-M0039-D11GVPSRFRGSGYGTDFSFSISSLQS QQYDSVPIT FGQGTRVEIK EDIATYYC 807A-M0037-F10GVPDRFSGSKSGTSASLAISGLRS AAWDDSLSGVV FGGGTKLTVL EDEADYYC 807A-M0028-B06GIPDRFSGSGSGTDFTLTISRLDPE QQYAYGRSPGYP FGQGTRLEIK DFGVYYC 807A-M0046-F05GIPDRFSGSGSGTDFTLTISRLEPE QQYGSSPYT FGQGTKLEIR DFAVYYC 807A-M0041-F03GVPSRFSGSVSGTQFTLTISSLQP QHYYTYPYA FGQGTKLEIK DDFATYYC 807A-M0043-E08GVPDRFSASKSGNTASLTVSGLQ NSYAGSNSLI FGGGTKLTVL AEDEADYYC 807A-M0042-D05GVPARFSASASGPDFTLTISSLQS QQYGGSPWYT FGQGTKLEIK EDFAVYYC 807A-M0029-G10GVPSRFSGSGSGTDFTLIISDLQPE QQSYTTPFT FGPGTTVDIK DFATYYC 807A-M0046-G03GVPSRFSGSGSGTDFSLTISSLQP QQSYSSLFT FGPGTKVDIK EDFATYYC 807A-M0037-D06GVPSRFSGSGSGTDFTLTISSLQP QQADTFPWT FGQGTKVDIK EDFATYFC 807A-M0043-E07GVPSRFSGTGSGTDFTLTISSLQP QQSYSDPLT FGGGTKVEIK EDFATYYC 807A-M0027-E11GVPSRFSGRGSGTEFTLTISSLQP QQSFSSPWT FGQGTKVEIK EDLATYYC 807A-M0046-A11GVPSRFSGSGSGTDFTLTISSLQP QQSYSTPLT FGGGTKVEIK EDFATYYC 807A-M0041-E01GVPSRFSGSGSGTDFTLTISSLQP QQFEDLFSLT FGPGTRVDLK EDIATYYC 807A-M0044-B07GIPDRFSGSGSGTDFTLTINRLEPE QQYHTSSFT FGPGTKVDIK DFAVYYC 807A-M0028-B02GVPSTFYGSGYGREFTLTISSLQP LQYDSFPYT FGQGTKLEIK EDFATYYC 807A-M0039-E06GVPSRFSGSGSGTDFTLTISSLQP QQSYSSPGIT FGPGTKVEIK EDFASYFC 807A-M0040-A03GIPERFSGSNSGNTATLTISGTQT QAWGSSPVV FGGGTRLTVL MDEADYYC 807A-M0044-G07GVPARFSGSGSGTDFTLTINNLQP QQANSFPIT FGGGTKVEIN EDFATYYC 807A-M0044-E08GVPSRFSGSGSGTDFTLTISSLQP QQAYSFPIT FGQGTRLEIK EDFATYYC 807A-M0038-A09GIPERFSGSNSGNTATLTISRVEA QLWDSSSDHPI FGTGTKVTVL GDEADYYC 807A-M0037-C08GVPSRFSGSASGADFTLTISSLQP QQTYDTPFT FGPGTTVDLK EDFATYYC 807A-M0039-H09GVPSRFSGSASGTNFSLTIDDLQP QQTFFSPPT FGQGTRVEIK EDFATYYC 807A-M0039-D05GVPSRFSGSGSGTEFTLTISSLQP QQHNNYPSFT FGPGTRLDIK EDFATYYC 807A-M0042-F12GVPDRFSGSKSGTSASLAISGLQS AAWDDSLNGWV FGGGTKLTVL EDEADYYC 807A-M0043-H05GVSTRFSGSKSGDTASLTISGLQT SSFTSRSTHV FGTGTKVTVL EDEADYYC 807A-M0042-C03GIPDRFSGSGSGTDFTLTISRLEPE QQSYSSPGIT FGPGTKVEIK DFASYFC 807A-M0040-C03GIPDRFSGSGSGTDFTLTISRLEPE QQSSSSPPT FGQGTRVEVR DFAVYWC 807A-M0046-C05GVPSRFSGSGSRTDFTLTISSLQP QQSYNTPPT FGQGTKLEIK EDFGIYYC 807A-M0027-D05GVPSRFSGSGSGTDFTLTISSLQP QQTYTTPAWT FGQGTKVEIR EDFATYYC 807A-M0040-B11GVPDRFSGSKSATSASLAISGLQS AAWDDSLNGPV FGGGTKLTVL EDEADYHC 807A-M0039-B02GVPSRFSAGGSGTEFTLTISSLQP QQYNSYSLT FGGGTKVEIK DDFGTYYC 807A-M0041-C07GVPSRFSGSGSGTDFTLIITNLQPD QQINGYPVT FGAGTKVEIK DFATYYC 807A-M0041-H04GVPDRFSGSKSGTSASLAITGLRS AAWDDSLSAVV FGGGTKLTVL EDETDYYC 807A-M0028-G07GIPERFSGSNSGDTATLTISWVEA HVWDSSSDHYV FGTGTAVTVL GDEAKYHC 807A-M0041-A09GIPARFSGSGSGTEFTLTINSLQSE QQYDNWPPFT FGPGTKVDIK DSAVYYC 807A-M0042-B10GISNRFSGSKSGNTASLTISGLQA SSYRSGSTYV FGTATKVTVL EDEADYYC 807A-M0041-E06GVPDRFSGSKSGTSASLAISGLQS AAWDDSLSGPV FGGGTKLTVL EDEADYYC 807A-M0037-D10GVPSRFSGSGSGTDFTLTIASLQP QQSYRLFPT FGQGTRLEIK DDFATYHC 807A-M0044-F04GVPARFSGSGSGTEFTLTISRMQP QQYSSWPA FGQGTMVEIN EDFVVYHC 807A-M0043-D10GIPARFSGSGSGTEFTLTISSLQSE QQYKDWPLT FGGGTQVEIK DFAVYYC 807A-M0043-G06GVPSRFSGSGSGTDFTLAISSLQP QNSYSSPYT FGQGTNVELK EDFATYYC 807A-M0037-G01GIPARFSGSGSGTEFTLTISSLQSE QQYGSSPPIT FGQGTRLEIK DFAVYYC 807A-M0044-E11GVSNRFSGSKSGNTASLTISGLQA SSYTNTITVV FGGGTKLTVL EDEADYYC 807A-M0043-A10GVPSRFSGSGSGTDFTLTISSLQP QQSYTIPTT FGGGTKVDVK EDFATYYC 807A-M0045-B03GVSDRFSGSKSGNTASLTISGLQA ASYTRTRSLA FGGGTRLTVL EDEADYHC 807A-M0038-A08GVPSRFSGSGTGTDFTLTIRSLQP QQSYSSPGIT FGPGTKVEIK EDFASYFC 807A-M0039-C02GIPDRFSGSGSATDFTLTISSLEPE QQYASPPRT FGQGTKVEIK DFAVYFC 807A-M0027-G01GVPDRFSGSKSGTSASLAISGLRS AAWDDSLSGPV FGGGTKLTVL EDEADYYC 807A-M0039-B08GVPSRFSGSGSGTDFTLTIDSLQP QQYYRYPLT FGGGTKVEIK EDFATYYC 807A-M0048-E12GVPSRFSGSGSGTEFTLTISSLQP LQHNSYPLT FGGGTKVEIK EDFATYYC 807A-M0041-A08GVSNRFSGSKSGNTASLTISGLQA SSYTSSSTLDPYA FGTGTKVTVL EDEADYYC807A-M0037-C09 GVPSRFSGSGSGTEFTLTISSLQP LQHNNYPYS FGQGTKLEIK EDFATYYC807A-M0040-G01 GVPSRFSGSGSATDFTLTISSLQP QQSFNTPFT FGPGTRVDIK EDFATYYC807A-M0045-E04 GVPSRFSGSGSGTDFTLTISSLQP LQDNDYPFT FGPGTKVEIR EDFATYYC807A-M0041-H05 GVPDRFSGSKSGTSASLAISGLQA QSYDSSLSGPI FGGGTTLTVL EDEAHYYC807A-M0043-A08 GVSNRFSTSQSANTASLTISGLQP SSYTTRSTWV FGGGTKLTVL EDEAEYFC807A-M0038-C09 GVPDRFSGSGSGTDFTLTISSLQA QQYYSTPTWT FGQGTKVEIK EDVAVYYC807A-M0042-F09 GVPSRFSGSGSGTEFTLTISSLQP LQHNSYPWT FGQGTKVEIK EDFATYYC807A-M0045-B12 GVPSRFSGSGFGTDFTLTISSLQP EQSYNVPRT FGQGTRLDIK EDFGIYYC807A-M0044-C04 GVPSRFSGSGSGTDFTLTITSLQP QQTYNTPGT FGQGTKLEIK EDFATYYC807A-M0026-F11 GVSNRFSGSKSGNTASLTISGLQA SSYTSSSTPYV FGTGTKVTVL EDEADYYC807A-M0027-E12 GIPDRFSGSGSGTDFTLTISRLEPE QQYVSSQLT FGGGTKVEIK DSALYYC807A-M0028-B12 GVPSRFSGSQSGTDFTLTISSLQP QQSFSPPIT FGQGTRLDIK EDFATYYC807A-M0026-G08 GVPSKFSGTGSGTEFTLTIGSLQP QQYFTFPLT FGGGTKVEIK EDSATYYC807A-M0043-A07 GVPSRFSGSGSGTDFTLTISSLQP QQANSFPLT FGGGTKVEIK EDFATYYC807A-M0042-F04 GIPARFSGSGSGTDFTLTISSLEPE QQANSFPPT FGQGTKVEIK DFATYYC807A-M0045-H09 GVPSRFSGSGSGTDFTLTISSLQP QQSYSIPPT FGQGTRVEIK EDFATYYC807A-M0046-D04 GIPDRFSGRGSGTDFSLTISRLEPE QQYEFSPEN FGQGTKLQIK DFAVYYC807A-M0040-G04 GVPSRFSGSGSGTDFTLTISSLQP QQSYSTPHT FGQGTKLEIK EDFATYYC807A-M0045-B01 GVSDRFSGSKSGNTASLTISGLQA SSYTNRNGYV FGTGTKVTVL DDETDYYC807A-M0040-A08 GVPDRFSGSKSGTSASLAISGLQS ASWDDSLNGVL FGGGTKLTVL EDDADYYC807A-M0026-F05 GVPVRFSGSGSGTDFTLTISSLGP QQGYNWPPWT FGQGTKVEIK EDFAVYYC807A-M0037-H02 GIPERFSGSGSGTDFTLTISRLEPE QQYGSSVA FGGGTKVEMK DFAVYYC807A-M0042-A06 GVPDRFSGSKSGTSASLAISGLRS AAWDDSLSGPV FGGGTKLTVL EDEADYYC807A-M0028-A07 GVPSRFSGSGSGTDFTLTISSLQP QKYNSAPRT FGQGTKVEIK EDVATYYC807A-M0043-G01 GVPSRFSGSGSGTEFTLTISSLQP QQYDSYWT FGQGTKIEIK DDFATYYC807A-M0046-F04 GVPSRFSGSGSETEFTLTISSLQP QQSYSAPLI FGGGTKVEIR EDFATYYC807A-M0037-F03 GIPARFSGSGSGTDFTLTISSLQPE QQSYEYPLT FGQGTKLEIK DFATYYC807A-M0039-C10 GVPDRFSGSKSGNTASLTISGLQA CSYAGSYTLL FGGGTKLTVL EDEADYYC

TABLE 10 Amino acid sequences of the VH chains of the antibodiesidentified using the screening strategy of Example 5 Initial Name FR1CDR1 FR2 CDR2 807A-M0027-C11 EVQLLESGGGLVQPGG EYGMS WVRQAPGKGLEWVSVISPSGGGTEYADSVKG SLRLSCAASGFTFS 807A-M0043-F08 EVQLLESGGGLVQPGG EYTMLWVRQAPGKGLEWVS GIWPSGGPTFYADSVKG SLRLSCAASGFTFS 807A-M0039-E07EVQLLESGGGLVQPGG FYFMG WVRQAPGKGLEWVS SISSSGGMTTYADSVKG SLRLSCAASGFTFS807A-M0039-D11 EVQLLESGGGLVQPGG FYGMG WVRQAPGKGLEWVS YISPSGGSTTYADSVKGSLRLSCAASGFTFS 807A-M0037-F10 EVQLLESGGGLVQPGG FYGMG WVRQAPGKGLEWVSYISSSGGLTFYADSVKG SLRLSCAASGFTFS 807A-M0028-B06 EVQLLESGGGLVQPGG FYQMNWVRQAPGKGLEWVS SIYPSGGLTYYADSVKG SLRLSCAASGFTFS 807A-M0046-F05EVQLLESGGGLVQPGG FYRMT WVRQAPGKGLEWVS SISSSGGGTPYADSVKG SLRLSCAASGFTFS807A-M0041-F03 EVQLLESGGGLVQPGG FYSMF WVRQAPGKGLEWVS YIYPSGGWTNYADSVKGSLRLSCAASGFTFS 807A-M0043-E08 EVQLLESGGGLVQPGG FYSMG WVRQAPGKGLEWVSYIYPSGGGTTYADSVKG SLRLSCAASGFTFS 807A-M0042-D05 EVQLLESGGGLVQPGG FYSMVWVRQAPGKGLEWVS SISPSGGQTDYADSVKG SLRLSCAASGFTFS 807A-M0029-G10EVQLLESGGGLVQPGG FYTMV WVRQAPGKGLEWVS VISPSGGLTHYADSVKG SLRLSCAASGFTFS807A-M0046-G03 EVQLLESGGGLVQPGG FYYMS WVRQAPGKGLEWVS RISPSGGLTHYADSVKGSLRLSCAASGFTFS 807A-M0037-D06 EVQLLESGGGLVQPGG HYLMV WVRQAPGKGLEWVSGISPSGGGTNYADSVKG SLRLSCAASGFTFS 807A-M0043-E07 EVQLLESGGGLVQPGG KYPMQWVRQAPGKGLEWVS SISPSGGSTVYADSVKG SLRLSCAASGFTFS 807A-M0027-E11EVQLLESGGGLVQPGG KYSMH WVRQAPGKGLEWVS GIYSSGGKTIYADSVKG SLRLSCAASGFTFS807A-M0046-A11 EVQLLESGGGLVQPGG LYGMV WVRQAPGKGLEWVS RISPSGGYTGYADSVKGSLRLSCAASGFTFS 807A-M0041-E01 EVQLLESGGGLVQPGG LYRMG WVRQAPGKGLEWVSSISPSGGWTRYADSVKG SLRLSCAASGFTFS 807A-M0044-B07 EVQLLESGGGLVQPGG MYGMLWVRQAPGKGLEWVS RISPSGGFTNYADSVKG SLRLSCAASGFTFS 807A-M0028-B02EVQLLESGGGLVQPGG MYMMD WVRQAPGKGLEWVS SIWPSGGQTWYADSVKG SLRLSCAASGFTFS807A-M0039-E06 EVQLLESGGGLVQPGG NYVMH WVRQAPGKGLEWVS VISPSGGATIYADSVKGSLRLSCAASGFTFS 807A-M0040-A03 EVQLLESGGGLVQPGG QYNMG WVRQAPGKGLEWVSYISSSGGITWYADSVKG SLRLSCAASGFTFS 807A-M0044-G07 EVQLLESGGGLVQPGG TYSMHWVRQAPGKGLEWVS YIGSSGGFTMYADSVKG SLRLSCAASGFTFS 807A-M0044-E08EVQLLESGGGLVQPGG TYWMI WVRQAPGKGLEWVS SISSSGGWTMYADSVKG SLRLSCAASGFTFS807A-M0038-A09 EVQLLESGGGLVQPGG VYSMA WVRQAPGKGLEWVS GIWPSGGPTAYADSVKGSLRLSCAASGFTFS 807A-M0037-C08 EVQLLESGGGLVQPGG WYAMD WVRQAPGKGLEWVSRIRPSGGNTDYADSVKG SLRLSCAASGFTFS 807A-M0039-H09 EVQLLESGGGLVQPGG WYAMDWVRQAPGKGLEWVS RIRSSGGLTHYADSVKG SLRLSCAASGFTFS 807A-M0039-D05EVQLLESGGGLVQPGG WYAMD WVRQAPGKGLEWVS SIYPSGGWTEYADSVKG SLRLSCAASGFTFS807A-M0042-F12 EVQLLESGGGLVQPGG WYAMS WVRQAPGKGLEWVS SIYSSGGKTGYADSVKGSLRLSCAASGFTFS 807A-M0043-H05 EVQLLESGGGLVQPGG WYAMV WVRQAPGKGLEWVSYIRSSGGHTVYADSVKG SLRLSCAASGFTFS 807A-M0046-H06 EVQLLESGGGLVQPGG WYDMDWVRQAPGKGLEWVS SISSSGGFTTYADSVKG SLRLSCAASGFTFS 807A-M0042-C03EVQLLESGGGLVQPGG WYDMD WVRQAPGKGLEWVS SIVSSGGLTDYADSVKG SLRLSCAASGFTFS807A-M0040-C03 EVQLLESGGGLVQPGG WYDME WVRQAPGKGLEWVS VIGPSGGPTHYADSVKGSLRLSCAASGFTFS 807A-M0046-C05 EVQLLESGGGLVQPGG WYDME WVRQAPGKGLEWVSWISSSGGTTWYADSVKG SLRLSCAASGFTFS 807A-M0027-D05 EVQLLESGGGLVQPGG WYDMFWVRQAPGKGLEWVS SIYSSGITYYADSVKG SLRLSCAASGFTFS 807A-M0040-B11EVQLLESGGGLVQPGG WYDMG WVRQAPGKGLEWVS SISPSGGSTTYADSVKG SLRLSCAASGFTFS807A-M0039-B02 EVQLLESGGGLVQPGG WYDMH WVRQAPGKGLEWVS SISPSGGLTYYADSVKGSLRLSCAASGFTFS 807A-M0041-C07 EVQLLESGGGLVQPGG WYDMH WVRQAPGKGLEWVSSISPSGGLTYYADSVKG SLRLSCAASGFTFS 807A-M0041-H04 EVQLLESGGGLVQPGG WYDMHWVRQAPGKGLEWVS SISSSGGDTTYADSVKG SLRLSCAASGFTFS 807A-M0028-G07EVQLLESGGGLVQPGG WYDMH WVRQAPGKGLEWVS YISPSGGWTGYADSVKG SLRLSCAASGFTFS807A-M0041-A09 EVQLLESGGGLVQPGG WYDMN WVRQAPGKGLEWVS RISPSGGSTRYADSVKGSLRLSCAASGFTFS 807A-M0042-B10 EVQLLESGGGLVQPGG WYDMN WVRQAPGKGLEWVSSIGSSGGETRYADSVKG SLRLSCAASGFTFS 807A-M0041-E06 EVQLLESGGGLVQPGG WYDMQWVRQAPGKGLEWVS SISSSGGLTTYADSVKG SLRLSCAASGFTFS 807A-M0037-D10EVQLLESGGGLVQPGG WYDMR WVRQAPGKGLEWVS SISSSGGRTVYADSVKG SLRLSCAASGFTFS807A-M0044-F04 EVQLLESGGGLVQPGG WYDMS WVRQAPGKGLEWVS RIGSSGGATQYADSVKGSLRLSCAASGFTFS 807A-M0043-D10 EVQLLESGGGLVQPGG WYDMS WVRQAPGKGLEWVSYIVPSGGVTLYADSVKG SLRLSCAASGFTFS 807A-M0043-G06 EVQLLESGGGLVQPGG WYDMVWVRQAPGKGLEWVS SIVSSGGETRYADSVKG SLRLSCAASGFTFS 807A-M0037-G01EVQLLESGGGLVQPGG WYDMV WVRQAPGKGLEWVS YIRPSGGITFYADSVKG SLRLSCAASGFTFS807A-M0044-E11 EVQLLESGGGLVQPGG WYDMV WVRQAPGKGLEWVS YIVPSGGETSYADSVKGSLRLSCAASGFTFS 807A-M0043-A10 EVQLLESGGGLVQPGG WYDMY WVRQAPGKGLEWVSGIGPSGGSTYYADSVKG SLRLSCAASGFTFS 807A-M0045-B03 EVQLLESGGGLVQPGG WYDMYWVRQAPGKGLEWVS SIRSSGGLTNYADSVKG SLRLSCAASGFTFS 807A-M0038-A08EVQLLESGGGLVQPGG WYDMY WVRQAPGKGLEWVS SISPSGGITAYADSVKG SLRLSCAASGFTFS807A-M0039-C02 EVQLLESGGGLVQPGG WYDMY WVRQAPGKGLEWVS SISPSGGITAYADSVKGSLRLSCAASGFTFS 807A-M0027-G01 EVQLLESGGGLVQPGG WYEMG WVRQAPGKGLEWVSSIGPSGGETIYADSVKG SLRLSCAASGFTFS 807A-M0039-B08 EVQLLESGGGLVQPGG WYEMGWVRQAPGKGLEWVS SIYSSGGQTVYADSVKG SLRLSCAASGFTFS 807A-M0046-E12EVQLLESGGGLVQPGG WYEMN WVRQAPGKGLEWVS RIYPSGGPTWYADSVKG SLRLSCAASGFTFS807A-M0041-A08 EVQLLESGGGLVQPGG WYFMH WVRQAPGKGLEWVS SIYPSGGTTEYADSVKGSLRLSCAASGFTFS 807A-M0037-C09 EVQLLESGGGLVQPGG WYGMG WVRQAPGKGLEWVSYISSSGGLTIYADSVKG SLRLSCAASGFTFS 807A-M0040-G01 EVQLLESGGGLVQPGG WYGMSWVRQAPGKGLEWVS RIVSSGGPTGYADSVKG SLRLSCAASGFTFS 807A-M0045-E04EVQLLESGGGLVQPGG WYGMV WVRQAPGKGLEWVS SIYPSGGTTQYADSVKG SLRLSCAASGFTFS807A-M0041-H05 EVQLLESGGGLVQPGG WYNMD WVRQAPGKGLEWVS SIGPSGGPTKYADSVKGSLRLSCAASGFTFS 807A-M0043-A08 EVQLLESGGGLVQPGG WYNMG WVRQAPGKGLEWVSYIGPSGGETYYADSVKG SLRLSCAASGFTFS 807A-M0038-C09 EVQLLESGGGLVQPGG WYNMQWVRQAPGKGLEWVS VISPSGGGTYYADSVKG SLRLSCAASGFTFS 807A-M0042-F09EVQLLESGGGLVQPGG WYNMV WVRQAPGKGLEWVS WISSSGGMTRYADSVKG SLRLSCAASGFTFS807A-M0045-B12 EVQLLESGGGLVQPGG WYPMY WVRQAPGKGLEWVS VIYPSGGHTKYADSVKGSLRLSCAASGFTFS 807A-M0044-C04 EVQLLESGGGLVQPGG WYQME WVRQAPGKGLEWVSSISSSGGTTDYADSVKG SLRLSCAASGFTFS 807A-M0026-F11 EVQLLESGGGLVQPGG WYQMSWVRQAPGKGLEWVS SISSSGGHTFYADSVKG SLRLSCAASGFTFS 807A-M0027-E12EVQLLESGGGLVQPGG WYRMT WVRQAPGKGLEWVS SISPSGGVTLYADSVKG SLRLSCAASGFTFS807A-M0028-B12 EVQLLESGGGLVQPGG WYSMG WVRQAPGKGLEWVS WISSSGGGTPYADSVKGSLRLSCAASGFTFS 807A-M0026-G08 EVQLLESGGGLVQPGG WYSMH WVRQAPGKGLEWVSSIRSSGGWTKYADSVKG SLRLSCAASGFTFS 807A-M0043-A07 EVQLLESGGGLVQPGG WYSMLWVRQAPGKGLEWVS YIYPSGGATFYADSVKG SLRLSCAASGFTFS 807A-M0042-F04EVQLLESGGGLVQPGG WYSMN WVRQAPGKGLEWVS GISSSGGMTHYADSVKG SLRLSCAASGFTFS807A-M0045-H09 EVQLLESGGGLVQPGG WYTMG WVRQAPGKGLEWVS SIVSSGGTPYADSVKGSLRLSCAASGFTFS 807A-M0046-D04 EVQLLESGGGLVQPGG WYTMH WVRQAPGKGLEWVSSIRSSGGMTDYADSVKG SLRLSCAASGFTFS 807A-M0040-G04 EVQLLESGGGLVQPGG WYTMQWVRQAPGKGLEWVS SIYPSGGDTKYADSVKG SLRLSCAASGFTFS 807A-M0045-B01EVQLLESGGGLVQPGG WYTMV WVRQAPGKGLEWVS SIRSSGGQTKYADSVKG SLRLSCAASGFTFS807A-M0040-A08 EVQLLESGGGLVQPGG WYTMV WVRQAPGKGLEWVS SIVPSGGDTHYADSVKGSLRLSCAASGFTFS 807A-M0026-F05 EVQLLESGGGLVQPGG YYAMQ WVRQAPGKGLEWVSSLYPSGGNTSYADSVKG SLRLSCAASGFTFS 807A-M0037-H02 EVQLLESGGGLVQPGG YYGMVWVRQAPGKGLEWVS RISPSGGMTDYADSVKG SLRLSCAASGFTFS 807A-M0042-A06EVQLLESGGGLVQPGG YYHMD WVRQAPGKGLEWVS SIVSSGGFTAYADSVKG SLRLSCAASGFTFS807A-M0028-A07 EVQLLESGGGLVQPGG YYRMA WVRQAPGKGLEWVS SIYSSGGMTLYADSVKGSLRLSCAASGFTFS 807A-M0043-G01 EVQLLESGGGLVQPGG YYSMI WVRQAPGKGLEWVSRISPSGGQTNYADSVKG SLRLSCAASGFTFS 807A-M0046-F04 EVQLLESGGGLVQPGG YYSMTWVRQAPGKGLEWVS SISPSGGGTGYADSVKG SLRLSCAASGFTFS 807A-M0037-F03EVQLLESGGGLVQPGG YYSMV WVRQAPGKGLEWVS WISSSGGSTNYADSVKG SLRLSCAASGFTFS807A-M0039-C10 EVQLLESGGGLVQPGG YYSMV WVRQAPGKGLEWVS WISSSGGSTNYADSVKGSLRLSCAASGFTFS Initial Name FR3 CDR3 FR4 807A-M0027-C11RFTISRDNSKNTLYLQMNS AVGYGDYGDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0043-F08RFTISRDNSKNTLYLQMNS ELDTAMAPPSDAFDI WGQGTMVTVSS LRAEDTAVYYCAR807A-M0039-E07 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR807A-M0039-D11 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR807A-M0037-F10 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR807A-M0028-B06 RFTISRDNSKNTLYLQMNS DRGVSLLGAFDI WGQGTMVTVSSLRAEDTAVYYCAR 807A-M0046-F05 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0041-F03 RFTISRDNSKNTLYLQMNS SVVGWGLDY WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0043-E08 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0042-D05 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0029-G10 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0046-G03 RFTISRDNSKNTLYLQMNS DFFTSYFDY WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0037-D06 RFTISRDNSKNTLYLQMNS GPGYSYGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAK 807A-M0043-E07 RFTISRDNSKNTLYLQMNSGVTTVPRYYYYYYMDV WGKGTTVTVSS LRAEDTAVYYCAR 807A-M0027-E11RFTISRDNPKNTLYLQMNS SLDLDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0046-A11RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0041-E01RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0044-B07RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0028-B02RFTISRDNSKNTLYLQMNS SVLLDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0039-E06RFTISRDNSKNTLYLQMNS DWGPFEAFDI WGQGTMVTVSS LRAEDTAVYYCAR 807A-M0040-A03RFTISRDNSKNTLYLQMNS DFFTSYFDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0044-G07RFTISRDNSKNTLYLQMNS GLYR WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0044-E08RFTISRDNSKNTLYLQMNS EDNYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR 807A-M0038-A09RFTISRDNSKNTLYLQMNS EDFWSGLEDV WGKGTTVTVSS LRAEDTAVYYCAR 807A-M0037-C08RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR807A-M0039-H09 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSSLRAEDTAVYYCAR 807A-M0039-D05 RFTISRDNSKNTLYLQMNS GLGMDV WGQGTTVTVSSLRAEDTAVYYCAR 807A-M0042-F12 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0043-H05 RFTISRDNSKNTLYLQMNSEPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR 807A-M0046-H06RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR807A-M0042-C03 RFTISRDNSKNTLYLQMNS QEVWQWPAQFDS WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0040-C03 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0046-C05 RFTISRDNSKNTLYLQMNSEPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR 807A-M0027-D05RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR807A-M0040-B11 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSSLRAEDTAVYYCAR 807A-M0039-B02 RFTISRDNSKNTLYLQMNS QEVWQWPAQFDSWGQGTLVTVSS LRAEDTAVYYCAR 807A-M0041-C07 RFTISRDNSKNTLYLQMNSQEVWQWPAQFDS WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0041-H04RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR807A-M0028-G07 RFTISRDNSKNTLYLQMNS QEVWQWPAQFDS WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0041-A09 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0042-B10 RFTISRDNSKNTLYLQMNSEPIWGYYYYGMDV WGQGTTVTVSS LKAEDTAVYYCAR 807A-M0041-E06RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR807A-M0037-D10 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSSLRAEDTAVYYCAR 807A-M0044-F04 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0043-D10 RFTISRDNSKNTLYLQMNSEPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR 807A-M0043-G06RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR807A-M0037-G01 RFTISRDNSKNTLYLQMNS QEVWQWPAQFDS WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0044-E11 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0043-A10 RFTISRDNSKNTLYLQMNSEPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR 807A-M0045-B03RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTLVTVSS LRAEDTAVYYCAR807A-M0038-A08 RFTISRDNSKNTLYLQMNS QEVWQWPAQFDS WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0039-C02 RFTISRDNSKNTLYLQMNS QEVWQWPAQFDSWGQGTLVTVSS LRAEDTAVYYCAR 807A-M0027-G01 RFTISRDNSKNTLYLQMNSDRGVSLLGAFDI WGQGTMVTVSS LRAEDTAVYYCAR 807A-M0039-B08RFTISRDNSKNTLYLQMNS QEVWQWPAQFDS WGQGTLVTVSS LRAEDTAVYYCAR807A-M0046-E12 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSSLRAEDTAVYYCAR 807A-M0041-A08 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0037-C09 RFTISRDNSKNTLYLQMNS DLWFGEWDYWGQGTLVTVSS LRAEDTAVYYCAR 807A-M0040-G01 RFTISRDNSKNTLYLQMNSEPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR 807A-M0045-E04RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0041-H05RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR807A-M0043-A08 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSSLRAEDTAVYYCAR 807A-M0038-C09 RFTISRDNSKNTLYLQMNS VKWDHSPLFDP WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0042-F09 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSSLRAEDTAVYYCAR 807A-M0045-B12 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0044-C04 RFTISRDNSKNTLYLQMNSEPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR 807A-M0026-F11RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0027-E12RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0028-B12RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR 807A-M0026-G08RFTISRDNSKNTLYLQMNS SSGIYYGYYMDV WGKGATVTVSS LRAEDTAVYYCAR807A-M0043-A07 RFTISRDNSKNTLYLQMNS GRSTFDI WGQGTMVTVSS LRAEDTAVYYCAR807A-M0042-F04 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTIVTVSSLRAEDTAVYYCAR 807A-M0045-H09 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0046-D04 RFTISRDNSKNTLYLQMNSSSGIYYGYYMDV WGKGATVTVSS LRAEDTAVYYCAR 807A-M0040-G04RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSS LRAEDTAVYYCAR807A-M0045-B01 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDV WGQGTTVTVSSLRAEDTAVYYCAR 807A-M0040-A08 RFTISRDNSKNTLYLQMNS EPIWGYYYYGMDVWGQGTTVTVSS LRAEDTAVYYCAR 807A-M0026-F05 RFTISRDNSKNTLYLQMNSGRGNYDFWSAGYYYY WGKGTTVTVSS LRAEDTAVYYCAR YMDV 807A-M0037-H02RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTPVTVSS LRAEDTAVYYCAR 807A-M0042-A06RFTISRDNSKNTLYLQMNS DRGVSLLGAFDI WGQGTMVTVSS LRAEDTAVYYCAR807A-M0028-A07 RFTISRDNSKNTLYLQMNS ESIAVAGVDY WGQGTLVTVSS LRAEDTAVYYCAR807A-M0043-G01 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR807A-M0046-F04 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR807A-M0037-F03 RFTISRDNSKNTLYLQMNS DLWFGEWDY WGQGTLVTVSS LRAEDTAVYYCAR807A-M0039-C10 RFTISRDNSKNTLYLQMNS DLWFREWDY WGQGTLVTVSS LRAEDTAVYYCAR

TABLE 11 Amino acid sequences of the VL chains of the antibodiesidentified using the screening strategies of Examples 21 and 22 AntibodyName VL-FR1 VL-CDR1 VL-FR2 VL-CDR2 VI-FR3 VL-CDR3 VL-FR4 807B-M0001-A09QSVLTQPPSASGTPGQ SGSSSNIGTYPVN WYQQLPGAAPKLLIY STDQRPSGVPDRFSGSKSGTSASLAISGLQSE AAWDDSLNGLWV FGGGTKVTVL RVTISC DESDYYC807B-M0001-B07 QYELTQPPSVSGTPGQ SGSSSNIGSEYVY WFQQLPGTAPRLLIY RNDQRPSGVPDRFSGSKSGTSASLAISGLQSE AAWDDSLPGWC SGGGTKLTVL RVTISC DEADYYC807B-M0001-C10 QYELTQPPSASGTPGQ SGSSSNIGTNFVY WYQQLPGTAPKLLIY RSIKRPSGVPDRFSGSKSGTSASLAISGLRSE AAWDDSLSGW FGGGTKLTVL TVTISC DEADYYC807B-M0001-G03 QSALTQPPSASGTPGQ SGSSSNIGINSVN WYQQLPGTAPKLLIY SNNQRPSGVPDRFSGSKSGTSASLAISGLRSE AAWDDSLAGWV FGGGTKVTVL RVTFSC DEADYYC8076-M0004-A03 QSELTQPPSASGTPGQ SGSSSNIGSNTVN WYQQLPGTAPKLLIY NNNQRPSGVPDRFSGSKSGTSASLAISGLQSE AAWHDGLNGPV FGGGTKLTVL RVTISC DEADYYC807B-M0004-A05 QDIQMTQSPATLSLSP KASQSVRAFIA WYQQKPGQAPRLLIS GASNRATGIPDRFSGGGSGTDFTLTISRLEPED QQYGSSRYT FGQGTKLEIK GERATLSC FAVYYC8076-M0004-B10 QDIQMTQSPSSLSASV RASQSISTYLN WYQQKPGKAPKLLIF ATSRLQSEVPSRFSGSGSGTDFTLTISSLQPE QQSYSPPRT FGQGTKLDIN GDRVTITC DFATYYC807B-M0004-C01 QDIQMTQSPLSLPVSLG RSSQSLVHTDGTTYLS WFQQRPGQSPRRLVYKVSDRGS GVPDRFSGSGSGIDFTLKISRVEAE MQGTHWPPT FGQGTKLEIK QKASISC DVGLYYC807B-M0004-C04 QDIQMTQSPSSLPVTP RSSQSLLHSSGYNYLD WYLQKPGQSPQLLIY LGSNRASGVPDRFTGSGSGTDFTLKISRVEAE MQALQTPT FGGGTKVDIK GEPASISC DVGVYYC807B-M0004-C05 QDIQMTQSPATLSVSP RASQSVSSNLA WYQQKPGQAPRLLIY GASTRATGVPARFSGSGSGTDFTLSISSLQPE QQYAGHPIT FGQGTRLEIK GERATLSC DFATYYC807B-M0004-D10 QSELTQPSSASETPGQ SGSSSNIGSNLVY WYQQVPGTALKLLIY RNDQRPSGVPDRFSGSKSGTSASLAISGLRSE VSWDGSLSGWV FGGGTRLTVL RVTISC DEADYFC807B-M0004-F06 QSELTQAASVSGSPGQ TGATRDVS WYQQHPGKAPKLVL YEVSSRPSGVSDRFSGSMSGNTASLTISGLQAE SSTTSRAPRW FGGGTKLTVL SITLSC DEADYYC807B-M0004-F07 QDIQMTQSPATLSLSP RASQSVSSYLA WYQQKPGQAPRLLIY DAFNRATGIPARFSGSGSGTDFTLTISSLEPED QQRSNWPLT FGGGTKVEIK GERATLSC FAVYYC807B-M0004-F10 QDIQMTQSPLSLPVTP RSSQSLMHRNGHHFFD WYLQKPGQSPQLLIY WASNRAPGVPDRFSGSGSGTDFTLKISRVEAE MQALQTPYT FGQGTKLEIK GEPASISC DVGIYYC807B-M0004-G08 QDIVMTQTPPSLPVNP RSSQSLVHSDGNTYLN WFQQRPGQSPRRLIS KVSNRDSGVPDRFSGSGAGTDFTLNISRVEAE MQVTEFPLT FGGGTKVEIK GEPASISC DVGDYYC807B-M0004-H03 QDIQMTQSPSSLSASIG QASQNIDNYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPE QQSYSTPRT FGQGTKVEIK DRVTISC DFATYYC807B-M0008-A03 QSALTQPASVSGSPGQ TGTSNDVGGYNYVS WYQQHPGIAPKVVIY EVSNRPSGVSDRFSGSKAGNTASLTISGLQAE NSYTSSRTWV FGGGTKVTVL SITISC DEADYYC807B-M0008-A08 QDIQMTQSPSSLSASV RASQSISSYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPE QQSYSTPLT FGGGTKVEIK GDRVTITC DFATYYC807B-M0008-B04 QYELTQPPSASGTPGQ SGTLSNIGTNIVS WFQQLPGTAPKLLIY NDHRRPSGVPDRFSGSKSATSASLAISGLQSE AAWDDSLNGW FGGGTKLTVL RVTISC DEADYYC807B-M0008-B08 QDIQMTQSPSSLSASV RASQSISTYLN WYQEKPGKAPELLIF AASSLQGGVPSRFSGSGSGTDFTLTISSLQPE QQSYDIPLS FGGGTKVEIK GDRVTITC DLATYYC807B-M0008-D02 QDIQMTQSPATLSLSLG RASEYISTYLA WYQQKPGQAPRLLIY DASVRAPGTPARFSGTGSGTDFTLTISSLQPD QERYDWPLT FGPGTRLDVK ERANFSC DFAVYFC807B-M0008-D05 QSALTOPPSASGTPGQ SGSSSNIGRNFVY WYRQLPGTAPKLLIY ENNQRPSGVPDRFSGSKSGTSASLAISGLRSE AAWDDSLSGLV FGGGTKLTVL RVTISC DEADYHC807B-M0008-E01 QDIQMTQSPGTLSLSP RASQSVSSRYLA WYQQKPGQAPRLLIY GASSRAPGIPDRFSGSGSGTDFTLTISSLEPEE QQYGSSPVT FGGGTKVEIK GERGTLSC SAVYYC807B-M0008-E06 QYELTQPPSASGTPGQ SGSSSNIGSNYVY WHQQLPGTAPKLLIS RNNQRPSGVPDRFSGSKSGTSASLAISGLRSE AAWDDSLSGW FGAGTKVTVL RVTISC DEADYYC807B-M0008-G11 QSVLTQPASVSGSPGQ TGASSDVGGSNFVS WYQQHPGKAPKLIIY DVSNRPSGVSNRFSGSKSGNTASLTISGLQAE SSYTSSSLW FGGGTKLTVL SITISC DDDDTDYYC807B-M0009-A06 QSVLTQPPSASGTPGQ SGSSSNIGSYYVY WYQHLPGTAPKLLIY RNNQRPSGVPDRFSGSKSGTSASLAISGLRSE AAWDDRLSTWV FGGGTKLTVL RVSISC DESDYYC807B-M0009-A09 QYELTQPPSVSVSPGQ SGDKLGDKYAS WYQQKPDQSPVLVIY QDRKRPSGIPERFSGSNSGNTATLTISGTQAM QAWDSNTVV FGGGTKLTVL TASITC DEADYYC807B-M0009-B07 QDIQMTQSPGTLSLSP RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLEPED QQYGSSGWT FGQGTKVEIK GERATLSC FAVYYC807B-M0009-C02 QYELTQPPSVSVSPGQ SGDKLGDKYTS WHQQKPGQSPVLVIY QDRKRPSGIPERFSGSNSGNTATLTISGTQAM QAWDSNTVV FGGGTKLTVL TASITC DEADYYC807B-M0009-C03 QSELTQPPSASGTPGQ SGSSSNIGSNYVY WYQQLPGTAPKLLIY RNNQRPSGVPDRFSGSKSGTSASLAISGLQSE AAWDDSLNAWV FGGGTKLTVL RVTISC DEADYYC807B-M0009-F06 QDIQMTQSPLSLPVTP KSSQSLLHSNGYNYLD WYLQKPGQSPQLLIS LGSNRASGVPARFSGSGSGTDFTLKISRVEAE MQALQTIT FGQGTRLEIK GEPASISC DVGVYYC807B-M0009-G03 QSVLTQSPSASASLGA TLSSGHSNYDIA WHQQQPEKGPRY LMKLNSDGIPDRFSGSSSGTERYLTISSLQSED QTWGTGLRV FGGGTKLTVL SVRVTC EADYYC807B-M0023-C03 QSELTQPPSVSVSPGQ SGYDLGAKFVS WYQQKSGQSPVLVM YQDNKRPSGIPERFSGSNSGNTATLTISGTQAM QVWDSPSYI FGTGTTVTVL TATITC DEADYYC807B-M0023-G05 QDIQMTQSPGTLSLSP RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISSLHPED QQGNSFPIT FGQGTRLEIK GERATLSC FATYFC807B-M0024-H08 QDIQMTQSPSSLSASV RASQSISSHLN WYQQKPGKVPKVLIY GASRLQSGVPSRFSGSGSGTDFTLTINSLQPE QQSYRAPVFT FGPGTKVDVK GDRVSITC DFATYYC807B-M0025-B05 QSVLTQPPSASGTPGQ SGSSSNIGRNPVN WYQHLPGTAPKLLIY GDNQRPSGVPDRFSGSRSGTSASLAISGLQSE AAWDDSLYGPV FGGGTKLTVL RVTISC DEADVYC807B-M0027-E08 QSALTQPPSVSVSPGQ AGDELGNKYAS WYQQKPGQSPVLVIY QDRKRPSGIPERFSGSHSGNTATLTISGTQALD QSWDSSSVI FGGGTKVTVL TASITC EADYYC807B-M0042-A05 QDIQMTQSPSAMSASV RASQGISNYLA WFQQKPGKVPKRLIY AASSLQSGVPSRFSGSGSGTEFTLTISSLQPE LQHNSYPLT FGGGTKVEIK GDRVTITC DFATYYC807B-M0042-B05 QSALTQPPSASGTPGQ SGSSSNIGSHYVY WYQQLPGTDPKLLIY KSIQRPSGVPDRFSGSKSGTSASLAISGLRSD AAWDDSLSGSYV FGTGTKVTVL RVTISC DEGDYYC807B-M0046-E03 QDIQMTQSPSSVSASV RASQDISSWLA WYQQKPGKAPKLLIY AASRLQSGVPSRFSGSGSGTDFSLTISSLQPD QQSHSFPLS FGGGTKVEIK GDRVTLTC DFATYYC807B-M0050-A04 QDIQMTQSPSSLSVSP RASQTISNDLA WYQQTPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISNLQPED QQADSFPLT FGGGTKVEIK GERATLSC FATYFC807B-M0050-B09 QDIQMTOSPDSLAVSL QSSQSVLYSSNNKNYLA WYQQKPGQPPKLLIYGASTRES GVPDRFSGSGSGTDFTLTISSLQAE QQYYTTPLT FGGGTKVEIK GERATINC DVAVYYC807B-M0050-E04 QDIQMTQSPSSVSASV RASQGISSWLA WYQQKPGKAPKLLIY PASSLQSGVPSRFSGSGSGTDFTLTISSLQPE QQGTSFPLT FGGGTKVEIK GDRVTITC DFATYYC807B-M0050-G01 QDIQMTQSPSTLSASV RASQSISTWLA WYQQKPGKAPKLLIY KAFSLEGGVPSRFSGSGSGTEFTLTISSLQPE QQSYSPPLT FGGGTKVDIR GDRVTITC DFATYYC807B-M0050-G05 QDIQMTQSPSSLSASV RASOGISNYLA WYQQKPGKVPKLLIY AASTLQSGVPSRFSGSGSGTDFTLTISSLQPE QNYNRAPRT FGQGTKVEIK GDRVTITC DVATYYC807B-M0050-H05 QDIQMTQSPPSVSASV RASQVITRWLA WYQQKPGQAPKLLIY SASSLQSGVPSRFSGSGSGTDFTLTISSLQPE QQATSFPLT FGGGTKVEIK GDRVTITC DFATYYC807B-M0050-H10 QDIQMTQSPLSLSASV RASQSISSYLN WYQHKPGKAPRLLIY GASSLQNGVPSRFTGSGTGTDFTLTISSLQPE QQSFTTPLT FGGGTKVEIK GDRVTITC DFATYYC

TABLE 12 Amino acid sequences of the VH chains of the antibodiesidentified using the screening strategies of Examples 21 and 22 IsolateName VH-FR1 VH-CDR1 VH-FR2 VH-CDR2 VH-FR3 VH-CDR3 VH-FR4 807B-M0001-A09EVQLLESGGGLVQPGGSLR RYPMF WVRQAPGKGLEW SISSSGGYTVYADSVKGRFTISRDNSKNTLYLQMNSLRAE VGKGAYYYAMDV WGQGTTVTVSS LSCAASGFTFS VSDTAVYYCAR 807B-M0001-B07 EVQLLESGGGLVQPGGSLR FYGMV WVRQAPGKGLEWSISPSGGYTLYADSVKG RFTISRDNSKNTLYLQMNSLRAE DGRRPHYGSGRWAY WGQGTLVTVSSLSCAASGFTFS VS DTAVYYCAK 807B-M0001-C10 EVQLLESGGGLVQPGGSLR QYVMFWVRQAPGKGLEW SISSSGGKTSYADSVKG RFTISRDNSKNTLYLQMNSLRAERLKIYDSSGYYYYYGMDV WGQGTTVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0001-G03EVQLLESGGGLVQPGGSLR SYAMS WVRQAPGKGLEW SISPSGGFTPYADSVKGRFTISRDNSKNTLYLQMNSLRAE VGKGAYYYAMDV WGQGTTVTVSS LSCAASGFTFS VSDTAVYYCAR 807B-M0004-A03 EVQLLESGGGLVQPGGSLR RYLMM WVRQAPGKGLEWVISPSGGRTWYADSV RFTISRDNSKNTLYLQMNSLRAE SIAAAGTDY WGQGTLVTVSSLSCAASGFTFS VS KG DTAVYYCVR 807B-M0004-A05 EVQLLESGGGLVQPGGSLR NYFMIWVRQAPGKGLEW WISPSGGTTQYADSV RFTISRDNSKNTLYLQMNSLRAE EAGY WGQGTLVTVSSLSCAASGFTFS VS KG DTAVYYCAR 807B-M0004-B10 EVQLLESGGGLVQPGGSLR KYVMIWVRQAPGKGLEW SISPSGGDTTYADSVKG RFTISRDNSKNTLYLQMNSLRAE DLGSNWGTGVVWNWGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0004-C01 EVQLLESGGGLVQPGGSLRNYNMH WVRQAPGKGLEW SIYSSGGTTLYADSVKG RFTISRDNSKNTLYLQMNSLRAELVADEWIDAFDI WGQGTMVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0004-C04EVQLLESGGGLVQPGGSLR AYYMG WVRQAPGKGLEW VIRPSGGKTKYADSVKGRFTISRDNSKNTLYLQMNSLRAE GPHGQGGVDS WGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR807B-M0004-C05 EVQLLESGGGLVQPGGSLR EYFMT WVRQAPGKGLEW SIRPSGGKTRYADSVKGRFTISRDNSKNTLYLQMNSLRAE VSRYYNNGAYRLDAFDI WGPGTVVTVSS LSCAASGFTFS VSDTAVYYCAR 807B-M0004-D10 EVQLLESGGGLVQPGGSLR WYQMS WVRQAPGKGLEWVISPSGGRTIYADSVKG RFTISRDNSKNTLYLQMNSLRAE QGLLTAFDI WGQGTMVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0004-F06 EVQLLESGGGLVQPGGSLR AYRMAWVRQAPGKGLEW YISSSGGVTSYADSVKG RFTISRDNSKNTLYLQMKSLRAE GTHLPGVDYWGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0004-F07 EVQLLESGGGLVQPGGSLREYYMT WVRQAPGKGLEW SIRPSGGATRYADSVKG RFTISRDNSKNTLYLQMNSLRAEEPIWGYYYYGMDV WGQGTTVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0004-F10EVQLLESGGGLVQPGGSLR GYIMA WVRQAPGKGLEW GIGSSGGLTAYADSVKGRFTISRDNSKNTLYLQMNSLRAE EAGY WGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR807B-M0004-G08 EVQLLESGGGLVQPGGSLR TYAMA WVRQAPGKGLEW SIRSSGGVTKYADSVKGRFTISRDNSKNTLYLQMNSLRAE GGAVAGY WGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR807B-M0004-H03 EVQLLESGGGLVQPGGSLR SYPMV WVRQAPGKGLEW GIWSSGGLTYYADSVRFTISRDNSKNTLYLQMNSLRAE EGSAGVVKGPARYYYYY WGKGTTVTVSS LSCAASGFTFS VS KGDTAVYYCAR MDV 807B-M0008-A03 EVQLLESGGGLVQPGGSLR HYTMY WVRQAPGKGLEWGISPSGGVTPYADSVKG RFTISRDNSKNTLYLQMNSLRAE AGSGGSFDY WGQGTLVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0008-A08 EVQLLESGGGLVQPGGSLR KYLMMWVRQAPGKGLEW YIWPSGGTDYADSVKG RFTISRDNSKNTLYLQMNSLRAE VRTSRINGSGFDYWGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAK 807B-M0008-B04 EVQLLESGGGLVQPGGSLRRYPMS WVRQAPGKGLEW SISPSGGPTSYADSVKG RFTISRDNSKNTLYLQMNSLRAE SGPYYFDYWGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0008-B08 EVQLLESGGGLVQPGGSLRHYPMS WVRQAPGKGLEW SISPSGGFTMYADSVKG RFTISRDNSKNTLYLQMNSLRAEFEYSSSSGISWFDP WGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0008-D02EVQLLESGGGLVQPGGSLR KYGMT WVRQAPGKGLEW SIRPSGGITKYADSVKGRFTISRDNSKNTLYLQMNSLRAE ENYGPDY WGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR807B-M0008-D05 EVQLLESGGGLVQPGGSLR TYHMF WVRQAPGKGLEW GISSSGGSTRYADSVKGRFTISRDNSKNTLYLQMNSLRAE VSVTTNAFDI WGQGTMVTVSS LSCAASGFTFS VS DTAVYYCAR807B-M0008-E01 EVQLLESGGGLVQPGGSLR TYAMT WVRQAPGKGLEW SISSSGGGTKYADSVKGRFTISRDNSKNTLYLQMNSLRAE HGYSSGWPPFDY WGQGTLVTVSS LSCAASGFTFS VSDTAVYYCAR 807B-M0008-E06 EVQLLESGGGLVQPGGSLR HYPMS WVRQAPGKGLEWSIVPSGGYTLYADSVKG RFTISRDNSKNTLYLQMNSLRAE HNRAIGTFDY WGQGTLVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0008-G11 EVQLLESGGGLVQPGGSLR SYAMIWVRQAPGKGLEW GISPSGGQTVYADSV RFTISRDNSKNTLYLQMNSLRAE AGSGGSFDYWGQGTLVTVSS LSCAASGFTFS VS KG DTAVYYCAR 807B-M0009-A06EVQLLESGGGLVQPGGSLR VYNMV WVRQAPGKGLEW VISPSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE DRGYCSGNTCYIDAFDI WGQGTMVTVSS LSCAASGFTFS VSDTAVYYCAR 807B-M0009-A09 EVQLLESGGGLVQPGGSLR RYPMA WVRQAPGKGLEWGISSSGGLTSYADSVKG RFTISRDNSKNTLYLQMNSLRAE FVGAKPADY WGQGTLVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0009-B07 EVQLLESGGGLVQPGGSLR FYWMVWVRQAPGKGLEW GISPSGGPTKYADSVKG RFTISRDNSKNTLYLQMNSLRAE EPIWGYYYYGMDVWGQGTTVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0009-C02 EVQLLESGGGLVQPGGSLRSYPMT WVRQAPGKGLEW GISSSGGSTAYADSVKG RFTISRDNSKNTLYLQMNSLRAE VTGGDFDYWGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAK 807B-M0009-C03 EVQLLESGGGLVQPGGSLRKYQMT WVRQAPGKGLEW VISSSGGDTAYADSVKG RFTISRDNSKNTLYLQMNSLRAEDRGYCSGNTCYIDAFDI WGQGTMVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0009-F06EVQLLESGGGLVQPGGSLR PYWMF WVRQAPGKGLEW GIVSSGGMTGYADSVRFTISRDNSKNTLYLQMNSLRAE VGMSTYAFDI WGQGTMVTVSS LSCAASGFTFS VS KGDTAVYYCAR 807B-M0009-G03 EVQLLESGGGLVQPGGSLR PYRMA WVRQAPGKGLEWSISPSGGHTGYADSVKG RFTISRDNSKNTLYLQMNSLRAE ESDGTTSAYFDY WGQGTLVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0023-C03 EVQLLESGGGLVQPGGSLR SYMMGWVRQAPGKGLEW YIYPSGGWTYYADSV RFTISRDNSKNTLYLQMNSLRAE GRSWGRYFQHWGQGTLVTVSS LSCAASGFTFS VS KG DTAVYYCAR 807B-M0023-G05EVQLLESGGGLVQPGGSLR MYWMG WVRQAPGKGLEW SISPSGGFTMYADSVKGRFTISRDNSKNTLYLQMNSLRAE GLYR WGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR807B-M0024-H08 EVQLLESGGGLVQPGGSLR AYNMD WVRQAPGKGLEW SIYPSGGHTNYADSVKGRFTISRDNSKNTLYLQMNSLRAE GKRIAARGGYYFDY WGQGTLVTVSS LSCAASGFTFS VSDTAVYYCAR 807B-M0025-B05 EVQLLESGGGLVQPGGSLR NYSMV WVRQAPGKGLEWSIVPSGGFTLYADSVKG RFTISRDNSKNTLYLQMNSLRAE HGSSWTFDY WGQGTLVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0027-E08 EVQLLESGGGLVQPGGSLR NYRMEWVRQAPGKGLEW SIWSSGGLTKqADSVKG RFTISRDNSKNTLYLQMNSLRAE GLYR WGQGTLVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0042-A05 EVQLLESGGGLVQPGGSLR HYQMKWVRQAPGKGLEW SIGSSGGSTSYADSVKG RFTISRDNSKNTLYLQMNSLRAE GPL WGQGTLVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0042-B05 EVQLLESGGGLVQPGGSLR NYHMDWVRQAPGKGLEW SISPSGGITKYADSVKG RFTISRDNSKNTLYLQMNSLRAE GVGATAGIWGQGTMVTVSS LSCAASGFTFS VS DTSVYYCAG 807B-M0046-E03 EVQLLESGGGLVQPGGSLRPYAMI WVRQAPGKGLEW YISPSGGKTQYADSVKG RFTISRDNSKNTLYLQMNSLRAE DQQTDYFDYWGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAK 807B-M0050-A04 EVQLLESGGGLVQPGGSLRQYNMN WVRQAPGKGLEW GISSSGGPTVYADSVKG RFTISRDNSKNTLYLQMNSLRAEFRCTSTSCFSDY WGQGTLVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0050-B09EVQLLESGGGLVQPGGSLR NYGML WVRQAPGKGLEW VISSSGGYTFYADSVKGRFTISRDNSKNSLYLQMNSLRAE VGATGPFDI WGQGTMVTVSS LSCAASGFTFS VS DTAVYYCAR807B-M0050-E04 EVQLLESGGGLVQPGGSLR RYSMM WVRQAPGKGLEW GISPSGGPTSYADSVKGRFTISRDNSKNTLYLQMNSLRAE ENIEYSSSFPNMGRPHYY WGQGTTVTVSS LSCAASGFTFS VSDTAVYYCAR YYYGMDV 807B-M0050-G01 EVQLLESGGGLVQPGGSLR RYEMY WVRQAPGKGLEWVISSSGGTTFYADSVKG RFTISRDNSKNTLYLQMNSLRAE GGDNWNYLSV WGQGTTVTVSSLSCAASGFTFS VS DTAVYYCAR 807B-M0050-G05 EVQLLESGGGLVQPGGSLR PYPMDWVRQAPGKGLEW VISSSGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAE VGMSTYAFDIWGQGTMVTVSS LSCAASGFTFS VS DTAVYYCAR 807B-M0050-H05 EVQLLESGGGLVQPGGSLRNYVMH WVRQAPGKGLEW SIGPSGGGTEYADSV RFTISRDNTKNTLYLQMNSLRAEDRGYCSGNTCYIDAFDI WGQGTMVTVSS LSCAASGFTFS VS KG DTAVYYCAR 807B-M0050-H10EVQLLESGGGLVQPGGSLR TYPMQ WVRQAPGKGLEW VISSSGGYTQYADSVKGRFTISRDNSKNTLYLQMNSLRAE MRVDYGDNYGMDV WGQGTTVTVSS LSCAASGFTFS VSDTAVYYCAS

TABLE 13 Enrichment Peptide Mapping Isolate Name Peptide VH CDR3 1 2 3 45 6 7 8 9 10 bCTD Fab IHC Group 807B-MO001-A09 E22-P1 6 8 +807B-MO001-B07 E22-P1 2 2 + + + +/− 4 807B-MO001-C10 E22-P1 1 1 +807B-MO001-G03 E22-P1 2 8 + 807B-MO042-A05 E22-P2 1 1 + 807B-MO042-B05E22-P2 7 7 + 807B-MO050-A04 E22-P3 19 19 + 807B-MO050-B07 E22-P3 2020 + + 807B-MO050-B09 E22-P3 4 4 + 807B-MO050-E04 E22-P3 1 1 +807B-MO050-G01 E22-P3 2 2 + 807B-MO050-G05 E22-P3 2 3 + 807B-MO050-H05E22-P3 1 8 + 807B-MO050-H10 E22-P3 1 1 + 807B-MO004-A03 E22-P4 55 + + + + 5 807B-MO004-A05 E22-P4 2 3 + +/− 1 807B-MO004-B10 E22-P4 33 + 807B-MO004-C01 E22-P4 2 2 + 807B-MO004-C04 E22-P4 1 1 + +/− 1807B-MO004-C05 E22-P4 1 1 + +/− 1 807B-MO004-D10 E22-P4 1 1 +807B-MO004-F06 E22-P4 2 2 + +/− 1 807B-MO004-F07 E22-P4 1 4 +807B-MO004-F10 E22-P4 1 3 + + +/− 2 807B-MO004-G08 E22-P4 1 1 +807B-MO004-H03 E22-P4 1 1 + + + 2 807B-MO023-C03 E23-P4 1 1 +807B-MO023-G05 E23-P4 1 149 + 807B-MO024-H08 E23-P4 4 4 + 807B-MO046-E03E22-P7 3 3 + 807B-MO008-A03 E22-P8 2 3 + 807B-MO008-A08 E22-P8 1 1 +807B-MO008-B04 E22-P8 2 2 + 807B-MO008-B08 E22-P8 1 1 + + 807B-MO008-D02E22-P8 5 5 + 807B-MO008-D05 E22-P8 2 2 + 807B-MO008-E01 E22-P8 2 2 +807B-MO008-E06 E22-P8 1 1 + 807B-MO008-G11 E22-P8 1 3 + 807B-MO008-G12E22-P8 1 1 + 807B-MO025-B05 E23-P8 1 1 + + 807B-MO027-E08 E23-P8 148149 + + + + 6 807B-MO009-A06 E22-P9 5 8 + + 807B-MO009-A09 E22-P9 1515 + 807B-MO009-B07 E22-P9 4 5 + + +/− + + + + + + + + 807B-MO009-C02E22-P9 13 13 + 807B-MO009-C03 E22-P9 2 8 + + + +/− 5 807B-MO009-F06E22-P9 1 3 + +/− 3 807B-MO009-G03 E22-P9 1 1 + +

TABLE 14 Amino acid sequences of the VL chains of the antibodiesidentified using the screening strategy of Example 23 Isolate Name FR1CDR1 FR2 CDR2 807B-M0011-C07 QYELTQPPSASGSP TGTSSDVGTYKYVSWYQQHPDKAPRLIIY EVNRRPS GQSVTISC 807B-M0012-C09 QSELTQPPSASGTPSGTLSNIGTNIVS WFQQLPGTAPKLLIY NDHRRPS GQRVTISC 807B-M0012-D09QDIQMTQSPATLSLS RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT PGERATLSC807B-M0012-F10 QDIQMTQSPSFLSA RASQGISNYLA WYQQKPGKAPKLLIY VASALQSSVGDRVTITC 807B-M0012-F12 QDIQMTQSPATLSV RASQSVSSNLA WYQQKPGQAPRLLIYAASSLQS SPGERATLSC 807B-M0012-G05 QDIQMTQSPGTLSL RASRSLFSTYLAWYQQKPGQPPRLLIY GASTRAT SPGERATLSC 807B-M0013-A12 QDIQMTQSPSTLSARASQSISRWLA WYQQKPGKAPKLLIY AASSLQS SVGDRVTITC 807B-M0013-B04QDIQMTQSPSSLPA RASQNIVTYLN WYQQKPGKAPKLLIY AASSLQS SVGDRVTITC807B-M0013-C03 QDIQMTQSPDSLSA RASQSISSYLN WYQQKPGKAPKLLIY AASSLQSSVGDRVTITC 807B-M0013-F06 QYELTQPPSASGTP SGSGSNIGSNLVY WYQQLPGTAPKLLIYRNTQRPS GQRVTISC 807B-M0013-G05 QDIQMTQSPGTLSL RASQSLSSSYLAWYQQKPGQPPRLLIY GASRRAT SPGERATLSC 807B-M0014-D07 QDIQMTQSPATLSLSRASQSVSRYVA WYQQKPGQSPRLVIY DASNRAT PGESTTLSC 807B-M0014-D09QDIQMTQSPSSFSA RASQGVGSYLA WYQQKPGKAPKLLIY GAYTLQS STGDRVTITC807B-M0014-E08 QDIQMTQSPSSLSA RASQDIRDDLG WYQQKPGKAPKRLIY AASSLQSSVGDRVTITC 807B-M0014-F07 QDIQMTQSPSSLSA RASQSIDTYLN WYQQKPGKAPKLLIYAASKLED SVGDRVAITC 807B-M0016-C06 QDIQMTQSPSSLSA RASQSINTYLNWYQQKPGQAPKLLIY ASSTLQR SVGDRVTITC 807B-M0016-D01 QDIQMTQSPSSLSARASQSIDNYLN WYQQKPGIAPKLLIY TASSLQS SVGDRVTITC 807B-M0016-D08QDIQMTQSPSSLSA RASQSISSFLN WYQQKPGKAPNLLIY GASSLQS SVGDRVAITC807B-M0016-E01 QDIQMTQSPSSLSA RASQSIGRYLN WYQQKPGKAPKLLIY TASSLQSSVGDRITITC 807B-M0016-F04 QDIQMTQSPATLSA RASQSVNNWVA WYQQKPGGAPEGLIYKASHLQS SVGDRVTFTC 807B-M0016-F05 QDIQMTQSPSSLSA RASQSISTYLNWYQQKPGKAPKLLIS APSRLQS SVGDSVTITC 807B-M0016-F08 QSALTQPRSVSGSPTGTSSDVGGYNYVS WYQQHPGKAPKLMIY DVSKRPS GQSVTISC 807B-M0017-B05QDIQMTQSPGTLSL RASQSVHSSYLA WYQQKPGQAPRLLIY GTSSRAT SPGERATLSC807B-M0017-B06 QYELTQPPSASGSP TGTSSDVGAYNYVS WYKIHPGKAPKLMIY DVNNRPSGQSVTISC 807B-M0017-E05 QDIQMTQSPGTLSL RASQSVTSSYLA WYQQKPGQAPRLLIYGASSRAT SPGERATLSC 807B-M0018-C12 QSELTQPPSASGSP TGSSRDIGNYNYVSWYQQFPGKAPKLIIY DVRKRPS GQTVTISC 807B-M0018-E09 QDIQMTQSPGTLSLRASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT SPGERATLSC 807B-M0018-G02QDIQMTQSPSSLSA RASQDIRDDLG WYQQKPGKAPKRLIY AASSLQS SIGDRVTITC807B-M0019-A04 QDIQMTQSPGTLSL RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRATSPGERATLSC 807B-M0019-A10 QDIQMTQSPLSLSAS RASQDIRSDLG WYQQKPGKAPKLLIYGASTLQS IGDRVTITC 807B-M0019-C01 QSALTQPASVSGSP SGTSSDVGGYNYVSWYQQHPGKAPKLVIY DVSNRPS GQPITISC 807B-M0019-F06 QSELTQPASVSGSPTGTSSDVGSYNLVS WYQQHPGKAPKLMIY EGSKRPS GQSITISC 807B-M0019-G07QDIQMTQSPATLSA RASQGLASWLA WYQQKPGKAPNLLIY KASNLKS SVGDRVTITC807B-M0020-D01 QDIQMTQSPSSLSA RASQTIRDYLH WYQQKPGKAPKLLIY AASSLQVSVGDRVSIT. 807B-M0020-F06 QDIQMTQSPGTLSL RASQSISYNYLA WYQQKPGQAPRLLIYDASNRAT SPGERATLSC 807B-M0020-F12 QDIQMTQSPSSLSA RASQDIRNYLAWFQQKPGKAPKSLIY GASSLQG SVGDRVTITC 807B-M0020-G01 QSELTQPASVSGSPTGTSSDVGSYNLVS WYQQHPGKAPKLMIY EGSKRPS GQSITISC 807B-M0079-B09QDIQMTQSPSSLSA RASQSISSYLN WYQQKPGNAPRLLIY SASTLNS SVGDRVTITC807B-M0079-D10 QSALTQPPSVSVSP AGDELGNKYAS WYQQKPGQSPVLVIY QDRKRPSGQTASITC 807B-M0079-H01 QDIQMTQSPSSLSA RASQSISSYLN WYQQKPGKAPKLLIYAASSLQS SVGDRVTITC 807B-M0079-H05 QDIQMTQSPSSLSA RASQGINSWLAWYQQRPGKAPRSLIY AATNLQN SVGDRVTITC 807B-M0080-A02 QDIQMTQSPLSLSASRASQSISTYLN WYQQKPGKAPKILIY AASSLQS VGDRVTIPC 807B-M0080-C04QSALTQPPSASGTP SGSTSNIGSNNVN WYQQLPGTAPKLLMY TTNYRPS GQTVAISC807B-M0080-F10 QDIQMTqSPSSVSAS RASQGISIWLA WYQQKPGKAPKLLIY GASSLQSIGDRVTITC 807B-M0081-C03 QDIQMTQSPSTLSA RASQSINRWLA WYQQKPGKAPKLLIYKASNLES SVGDRVTITC 807B-M0081-C05 QSALTQPPSVSGAP TGSSSNIGAPYDVHWYQQVPGTAPKVLIY GNNHRPS GQRVTISC 807B-M0081-D08 QDIQMTQSPSSVSARASQGISSWLA WYQQKPGKAPKLLIY AASSLQS SVGDRVTITC 807B-M0081-E08QDIQMTQSPSSLPA RASRNIGKYLN WYQQIRGRAPRLLVY LASSVQT SVGDRVTITC807B-M0081-F12 QDIQMTqSPSSLSAS RASQSISSYLN WYQQKPGKAPKLLIY AASSLQSVGDRVTITC 807B-M0081-G04 QDIQMTQSPDTLSLS RASQSISTSLA WYHQRPGQAPRLLIYDASNRAT PGERATLSC 807B-M0081-G11 QDIQMTQSPSSLSA RASQSIDTYLNWYQQKPGKAPKLLIY AASKLED SVGDRVAITC 807B-M0081-H03 QYELTQPPSASGSPTGTSSDVGGYNYVS WYQQHPGKAPKFMIY EVNKRPS GQSVTISC 807B-M0081-H07QDIQMTQSPSSLSA RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS SVGDRVTITC807B-M0082-B07 QSALTQPASVSGSP TGTSSDVGGYNYVS WYQQHPGKAPKLMIY DVSNRPSGQSITISC 807B-M0082-E01 QDIQMTQSPSSLSA RASQSIDTYLN WYQQKPGKAPKLLIYAASSLQS SVGDRVAITC 807B-M0082-E08 QDIQMTQSPSSLSA RASQSISSYLNWYQQKQGKAPKLLMF AASSLKS SIGDRVTITC 807B-M0082-H06 QDIQMTQSPSSLSARASQGIRNNLA WYQQRPGKAPKRLIY GASNLHS SVGDRVTVTC 807B-M0083-B10QDIQMTQSPSSVSA RASQDIHTWLA WYQQKPGKAPKLLIY AASSLQS SVGDRVTIIC807B-M0083-E10 QDIQMTQSPGTLSL RASQSISSRYLA WYQQKAGQAPRLLMY GASRATSPGERATLSC 807B-M0083-E11 QSALTQPPSVSVAP GGNNIGTKIVN WYQQRPGQAPVVVVYDNSDRPS GQTARITC 807B-M0084-C03 QYELTQPPSVSVAP GGSNIGSKSVHWYQQKSGQAPVLVVY DDSDRPS GQTARISC 807B-M0084-C11 QDIQMTQSPSSLSARASQSIATFLN WYQQKPGKAPNLLIS GAFNLQS SVGDRVTITC 807B-M0084-E07QDIQMTQSPSSLPA RASQSISRYLN WYQQKPGKAPKVMIY DASTLQS SVGDRVTITC807B-M0084-F03 QDIQMTQSPATLSV RASQSVSNNLA WYQQKPGQAPRLLIY AASTRATSPGERATLSC 807B-M0084-F08 QDIQMTQSPATLSV RASQSVRNNLA WYQQKPGQAPRLLIYGASTRAT SPGARATLSC 807B-M0084-H05 QSALTQPPSVSAAP SGGTSNIQYNGVNWYQQLPGKAPKLLIY FDDLLPS GQRVTISC 807B-M0085-B12 QDIQMTQSPSSVSARASQGISSWLA WYQQKPGKAPKLLIY AASSLQS SVGDRVTITC 807B-M0085-C01QDIQMTQSPATLSV RASQSVSSNLA WYQQKPGQAPRLLIY GASTRAT SPGERATLSC807B-M0085-E10 QDIQMTQSPSSLSA QASQDISKYLN WYQQRPGKAPELLIY DASNLEPSVGDRVTITC 807B-M0085-G03 QDIQMTQSPLSLSAS RASQSISSYLN WYQQKPGKAPKLLIYAASSLQS VGDRVTITC 807B-M0085-G07 QDIQMTQSPGTLSL RASQSVSSSYLAWYQQKPGQAPRLLIY GASSRAT SPGERATLSC 807B-M0085-G08 QDIQMTQSPGTLSLRASQSVSRSSLA WYQQKPGQAPRLLIY GASSRAT SPGERATLSC 807B-M0086-C06QSALTQPPSASGTP SGGSSNIGSNIVN WYQQVPGMAPKLLYT TNNRRPS GQKVTISC NNRRPS807B-M0086-D03 QDIQMTQSPSSLSA RASQSISSYLN WYQQKPGKAPKLLIY VASSLQSSVGDRVTITC 807B-M0086-E08 QDIQMTQSPGTLSL SVSQSVSSNYLA WYQQKPGQSPRLLIYGASARAT SPGERATLSC 807B-M0086-G03 QDIQMTqSPSFLSAS RASQVLRRPLAWYQQKAGKAPKLLIS AFSILES VGDRVSVTC Isolate Name FR3 CDR3 FR4807B-M0011-C07 GVPDRFSGSKSGNTASLTI YSHATGNNYV FGTGTKVTVL SGLQAEDEADYYC807B-M0012-C09 GVPDRFSGSKSATSASLAI AAWDDSLNGVV FGGGTKLTVL SGLQSEDEADYYC807B-M0012-D09 GIPARFSGSGSGTDFTLTI QQRSNWPRYT FGQGTKLEIK SSLEPEDFAVYYC807B-M0012-F10 GVPSRFSGSGSGTEFTLTI QQYYSYSA FGQGTRVEIK SSLQPEDFATYYC807B-M0012-F12 GVPSRFSGSGSGTDFTLT QQTYSTPWT FGQGTKLEIK VSSLQPEDFATYYC807B-M0012-G05 GIPDRFSGSGSGTDFTLTI QQYVSSQLT FGGGTKVEIK SRLEPEDSALYYC807B-M0013-A12 GVPSRFSGSGSGTDFTLTI QQSYSTPLT FGGGTKVEIK SSLQPEDFATYYC807B-M0013-B04 GVPSRFSGSGSGTDFTLTI QQSYSMSSWT FGQGTNLEIK SSLQPEDFATYYC807B-M0013-C03 GVPSRFSGSGSGTDFTLTI QQSYSTPPYT FGQGTKLEIK SSLQPEDFATYYC807B-M0013-F06 GVPDRFSASKSGTSASLAI ATWDDSLGGVV FGGGTKLTVL SGLRSEDEADYHC807B-M0013-G05 GIPDRFSGSGSGTDFTLTI QHYGRSPLFT FGPGTTVDIK SRLEPEDFAVYYC807B-M0014-D07 GIPARFSGSGSGTDFTLTIT LQRSNWPFT FGPGTKVEIK SLEPEDFGIYYC807B-M0014-D09 GVPSRFSGSGSGTDFTLII QQYYSYPFT FGPGTKVDIK SGLQSEDFATYYC807B-M0014-E08 GVPSRFSGSGSGTEFTLTI QQHNNYPSFT FGPGTRLDIK SSLQPEDFATYYC807B-M0014-F07 GVPSRFSGSGTGTDFTLTI QQSYSSPGIT FGPGTKVEIK RSLQPEDFASYFC807B-M0016-C06 GVPSRFSGSGSGTDFTLTI QQSYSPPLYT FGQGTKLDLK SSLQLEDFATYFC807B-M0016-D01 GVPSRFSGSGSGTDFTLTI QQSYTTPHT FGQGTKLEIR SSLQPEDFATYYC807B-M0016-D08 GVPSRFSGSGSGTDFTLTI QQSYSTPYT FGQGTKLEIK SSLQPEDFATYYC807B-M0016-E01 GVPSRFSGSGSGTDFTLTI QQSFTTPHT FGLGTKLEIE SSLQPEDFATYYC807B-M0016-F04 GVPSRFSGGGSGTVFTLTI QQYQTYPYT FGQGTRLDMK TSLQPDDFATYYC807B-M0016-F05 GVPSRFSDSGSGTDFTLAI QQSYSTPVT FGQGTKLEIK SSLQPEDFATYYC807B-M0016-F08 GVPDRFSGSKSGNTASLTI CSYAGNYSVV FGGGAKLSVL SGLQAEDEADYYC807B-M0017-B05 GIPDRFSGNGFGTDFTLTI QQYGSSPIT FGQGTRLEIK SRLEPEDFAVYYC807B-M0017-B06 GVSNRFSGSKSGNTASLTI CSYAGSSTQV FGTGTKVTVL SGLQAEDEADYYC807B-M0017-E05 GIPDRFSGSGSGADFTLTI QQYGTSPYT FGQGTKLEIK SRLEPEDFAVYYC807B-M0018-C12 GVSDRFSGSKSGNTAFLT GSYTGTNNV FGPGTSVTVL VSGLQTEDEADYFC807B-M0018-E09 GIPDRFSGSGSGTDFTLTI QQYGSSRVT FGGGTKVEIK SRLEPEDFAVYYC807B-M0018-G02 GVPSRFSGSGSGTEFTLTI LQHNTFPSFT FGPGTKVDIK SSLQPEDFATYYC807B-M0019-A04 GIPDRFSGSGSGTDFTLTI QQYGSSSIT FGQGTRLEIK SRLEPEDFAVYYC807B-M0019-A10 GVPSRFSGSGSGADFTLII LQDYNYPRT FGQGTKVEIK SNLQPEDFATYYC807B-M0019-C01 GISYRFSGSKSVNTASLTIS SSYTSNSTLV FGGGTQADRP GLQAEDEADYFC807B-M0019-F06 GVSNRFSGSKSGNTASLTI CSYAGSSTLV FGGGTKLTVL SGLQAEDEADYYC807B-M0019-G07 GVPSRFSGSESGTEFTLTI HQYYSNSWT FGQGTKVEIK SSLQPDDFATYFC807B-M0020-D01 GVPSRFSGSGSGTDFTLTI QQTYSTLIT FGQGTRLEIK SSLQPEDLATYYC807B-M0020-F06 GIPARFSGSGSGTDFTLTI QQRSNWPPGLT FGGGTKVEIK SSLEPEDFAVYYC807B-M0020-F12 GVPSKFSGSGSGTDFTLTI QQYNSYPLT FGGGTKVEVK SGLQPEDFATYYC807B-M0020-G01 GVSNRFSGSKSGNTASLTI CSYAGSSTYV FGTGTKVTVL SGLQAEDEADYYC807B-M0079-B09 GVPSRFSGSGSGTHFTLTI QQANSLPFT FGQGTKLEIK SSLQPEDFGIYYC807B-M0079-D10 GIPERFSGSHSGNTATLTI QSWDSSSVI FGGGTKVTVL SGTQALDEADYYC807B-M0079-H01 GVPSRFSGSGSGTDFTLTI QQSYSTPFT FGPGTKVDIK SSLQPEDFATYYC807B-M0079-H05 GVPSRFSGSGSGTLFTLTI QQYQNYPYT FGQGTKLDIE NNLQPEDFATYYC807B-M0080-A02 GVPSRFSGSGSGTDFTLTI QQSYTTPLT FGGGTKVEIK SSLQPEDFATYFC807B-M0080-C04 GVPARFSGSKSGTSASLAI AAWDDSLNGPNVV FGGGTKLTVLSGLQSEDEADYYC 807B-M0080-F10 GAPSRFSGSGSGTDFTLTI QQANSFPLT FGGGTKVEIKSSLQPEDFATYYC 807B-M0081-C03 GAPSRFSGSGSGTEFTLTI QQYHSYPWT FGQGTKVDVKSSLQPDDFGTYYC 807B-M0081-C05 GVPDRFSGSKSGTSASLAI QSYDSSLSGPI FGGGTTLTVLSGLQAEDEAHYYC 807B-M0081-D08 GVPSRFSGSGSGTDFTLTI QQANSFPPT FGQGTKVEIQSSLQPEDFATYYC 807B-M0081-E08 GVPPRFSGSGSGTDFSLII QQSYAAPLT FGGGTKVEIKSSLQPEDFATYYC 807B-M0081-F12 GVPSRFSGSGSGTEFSLSI QQANSFPLT FGGGTKVEIKSSLQPEDFATYYC 807B-M0081-G04 GVPARFSGTGSGTDFTLTI QQRSNWPYT FGQGTKLEIKSSLEPEDFAVYYC 807B-M0081-G11 GVPSRFSGSGTGTDFTLTI QQSYSSPGIT FGPGTKVEIKRSLQPEDFASYFC 807B-M0081-H03 GVPDRFSGSKSGNTASLT SSYAGRNFVV FGGGTKLTVLVSGLQAEDEADYYC 807B-M0081-H07 GVPSRFSGSGSGTDFTLII QQSYTTPFT FGPGTTVDIKSDLQPEDFATYYC 807B-M0082-B07 GVSNRFSGSKSGNTASLTI SSYTSRSTYV FGTGTKVTVLSGLQAEDEADYYC 807B-M0082-E01 GVPSRFSGSGSGTDFTLT LQSNTFPFT FGPGTKVDITVSSLQPEDFATYYC 807B-M0082-E08 GVPSRFSGSGSGTDFTLTI QQTYSSPWT FGQGTKVEIRSNLQPEDFATYYC 807B-M0082-H06 GVPSRFSGSGSGTEFTLTI LQHNNYPYS FGQGTKLEIKSSLQPEDFATYYC 807B-M0083-B10 GVPSRFSGSGSGTDFTLTI QQSYSTPRT FGQGTKVEIKSSLQPEDFATYYC 807B-M0083-E10 GIPARFSGSGSGTDFTLTI QQSYEYPLT FGQGTKLEIKSSLQPEDFATYYC 807B-M0083-E11 GIPERFSGSNSGNTATLTI QLWDSSSDHPI FGTGTKVTVLSRVEAGDEADYYC 807B-M0084-C03 GIPERFFGSNSGRTATLTIS QVWDSSSDDYV FAAGTKLSVLGVEVGDEADYYC 807B-M0084-C11 GVPSRFSGSGSGTDFTLTI QHSYGTPT FGQGTKVEIKSSLQPEDFATYYC 807B-M0084-E07 GVPSRFSGSGSGTDFTLTI QQSYITPRT FGQGTKVEIKSNLQPEDFATYYC 807B-M0084-F03 GIPARFSGSGSGTDFTLTI QQYYYTPPT FGRGTKVEINSSLEPEDFAVYYC 807B-M0084-F08 DIPARFSGGGSGTEFTLTI QHEET FGQGTKVEIKSSLQPDDFATYYC 807B-M0084-H05 GVSDRFSGSKSGTSASLAI AAWDDSLSGVV FGGGTKLTVLSGLRSEDEADYYC 807B-M0085-B12 GVPSRFSGSGSGTDFTLTI QQANSFPLT FGGGTKVEIKSSLQPEDFATYYC 807B-M0085-C01 GIPARFSGSGSGTDFTFTI QQYINLPIT FGQGTRLEIKSSLQPEDIATYFC 807B-M0085-E10 GVPSRFSGSGSGTHFTFTI QQFDNFPLT FGPGTRLDIKSSLQPEDFATYYC 807B-M0085-G03 GVPSRFSGSGSGTDFTLTI QQSYSTPLYT FGQGTKLEIKSSLQPEDFATYYC 807B-M0085-G07 GIPDRFSGSGSGTDFTLTI QSGVT FGGGTKVEIKSRLEPEDFAVYYC 807B-M0085-G08 GIPDRFSGSGSGTDFTLTI QQYGNSPGGT FGQGTKVEIKSRLEPEDFAVYYC 807B-M0086-C06 GVPDRFSGSKSGTSASLAI AAWDDSLSGGV FGGGTKLTVLSGLRSEDEADYYC 807B-M0086-D03 GVPSRFSGSGSGTDFTLTI QQSYSIPPT FGQGTRVEIKSSLQPEDFATYYC 807B-M0086-E08 GIPDRFSGSGSRTDFTLTI QQYVTTPPT FGQGTKVEIKSRLEPEDFAVYYC 807B-M0086-G03 GVPSRFSAGGSGTEFTLTI QQVSSYPLT FGGGPRVEIKNSLQAEDFATYYC

TABLE 15 Amino acid sequences of the VH chains of the antibodiesidentified using the screening strategy of Example 23 Isolate Name FR1CDR1 FR2 CDR2 807B-M0011-C07 EVQLLESGGGLVQPG WYSMD WVRQAPGKGLEWVSGIGPSGGRTRY GSLRLSCAASGFTFS ADSVKG 807B-M0012-C09 EVQLLESGGGLVQPG WYTMDWVRQAPGKGLEWVS GISPSGGATNY GSLRLSCAASGFTFS ADSVKG 807B-M0012-D09EVQLLESGGGLVQPG LYQMA WVRQAPGKGLEWVS SISSSGGLTDY GSLRLSCAASGFTFS ADSVKG807B-M0012-F10 EVQLLESGGGLVQPG WYNMS WVRQAPGKGLEWVS YIYPSGGITIYAGSLRLSCAASGFTFS DSVKG 807B-M0012-F12 EVQLLESGGGLVQPG WYDMDWVRQAPGKGLEWVS SIYPSGGLTGY GSLRLSCAASGFTFS ADSVKG 807B-M0012-G05EVQLLESGGGLVQPG WYRMT WVRQAPGKGLEWVS SISPSGGVTLYA GSLRLSCAASGFTFS DSVKG807B-M0013-A12 EVQLLESGGGLVQPG HYGMS WVRQAPGKGLEWVS SIRSSGGRTWYGSLRLSCAASGFTFS ADSVKG 807B-M0013-B04 EVQLLESGGGLVQPG HYDMVWVRQAPGKGLEWVS VIVPSGGATAY GSLRLSCAASGFTFS ADSVKG 807B-M0013-C03EVQLLESGGGLVQPG WYNMA WVRQAPGKGLEWVS SISPSGGHTKY GSLRLSCAASGFTFS ADSVKG807B-M0013-F06 EVQLLESGGGLVQPG KYVMT WVRQAPGKGLEWVS VISSSGGPTDYGSLRLSCAASGFTFS ADSVKG 807B-M0013-G05 EVQLLESGGGLVQPG WYSMIWVRQAPGKGLEWVS YIGPSGGPTRY GSLRLSCAASGFTFS ADSVKG 807B-M0014-D07EVQLLESGGGLVQPG WYTMT WVRQAPGKGLEWVS SISSSGGVTKY GSLRLSCAASGFTFS ADSVKG807B-M0014-D09 EVQLLESGGGLVQPG YYIMA WVRQAPGKGLEWVS SISPSGGGTVYGSLRLSCAASGFTFS ADSVKG 807B-M0014-E08 EVQLLESGGGLVQPG WYAMDWVRQAPGKGLEWVS SIYPSGGWTEY GSLRLSCAASGFTFS ADSVKG 807B-M0014-F07EVQLLESGGGLVQPG WYDMF WVRQAPGKGLEWVS SISPSGGFTQY GSLRLSCAASGFTFS ADSVKG807B-M0016-C06 EVQLLESGGGLVQPG LYGMS WVRQAPGKGLEWVS SIGPSGGHTFYGSLRLSCAASGFTFS ADSVKG 807B-M0016-D01 EVQLLESGGGLVQPG WYNMGWVRQAPGKGLEWVS GISPSGGTTTY GSLRLSCAASGFTFS ADSVKG 807B-M0016-D08EVQLLESGGGLVQPG WYGMV WVRQAPGKGLEWVS SIYPSGGTTQY GSLRLSCAASGFTFS ADSVKG807B-M0016-E01 EVQLLESGGGLVQPG WYDMQ WVRQAPGKGLEWVS SISSSGGLTTYAGSLRLSCAASGFTFS DSVKG 807B-M0016-F04 EVQLLESGGGLVQPG NYNMHWVRQAPGKGLEWVS VISPSGGGTWY GSLRLSCAASGFTFS ADSVKG 807B-M0016-F05EVQLLESGGGLVQPG WYRMT WVRQAPGKGLEWVS SISPSGGVTLYA GSLRLSCAASGFTFS DSVKG807B-M0016-F08 EVQLLESGGGLVQPG MYHMG WVRQAPGKGLEWVS GISPSGGTTTYGSLRLSCAASGFTFS ADSVKG 807B-M0017-B05 EVQLLESGGGLVQPG WYTMFWVRQAPGKGLEWVS GIWPSGGKTDY GSLRLSCAASGFTFS ADSVKG 807B-M0017-B06EVQLLESGGGLVQPG WYEMG WVRQAPGKGLEWVS SISPSGGYTSY GSLRLSCAASGFTFS ADSVKG807B-M0017-E05 EVQLLESGGGLVQPG WYQMA WVRQAPGKGLEWVS GISSSGGTTTYGSLRLSCAASGFTFS ADSVKG 807B-M0018-C12 EVQLLESGGGLVQPG FYRMGWVRQAPGKGLEWVS SISSSGGLTDY GSLRLSCAASGFTFS ADSVKG 807B-M0018-E09EVQLLESGGGLVQPG WYGMA WVRQAPGKGLEWVS YISPSGGGTSY GSLRLSCAASGFTFS ADSVKG807B-M0018-G02 EVQLLESGGGLVQPG KYVMN WVRQAPGKGLEWVS SISSSGGQTSYGSLRLSCAASGFTFS ADSVKG 807B-M0019-A04 EVQLLESGGGLVQPG YYDMWWVRQAPGKGLEWVS RIVSSGGWTMY GSLRLSCAASGFTFS ADSVKG 807B-M0019-A10EVQLLESGGGLVQPG YYDMS WVRQAPGKGLEWVS SIWSSGGNTMY GSLRLSCAASGFTFS ADSVKG807B-M0019-C01 EVQLLESGGGLVQPG WYGMG WVRQAPGKGLEWVS YISSSGGHTKYGSLRLSCAASGFTFS ADSVKG 807B-M0019-F06 EVQLLESGGGLVQPG WYPMDWVRQAPGKGLEWVS SISPSGGFTQY GSLRLSCAASGFTFS ADSVKG 807B-M0019-G07EVQLLESGGGLVQPG FYPMV WVRQAPGKGLEWVS WISSSGGTTSY GSLRLSCAASGFTFS ADSVKG807B-M0020-D01 EVQLLESGGGLVQPG WYVMT WVRQAPGKGLEWVS GISSSGGMTEYGSLRLSCAASGFTFS ADSVKG 807B-M0020-F06 EVQLLESGGGLVQPG VYNMYWVRQAPGKGLEWVS SISPSGGFTTYA GSLRLSCAASGFTFS DSVKG 807B-M0020-F12EVQLLESGGGLVQPG IYEMA WVRQAPGKGLEWVS SISPSGGWTKY GSLRLSCAASGFTFS ADSVKG807B-M0020-G01 EVQLLESGGGLVQPG FYYMS WVRQAPGKGLEWVS GISPSGGTTQYGSLRLSCAASGFTFS ADSVKG 807B-M0079-B09 EVQLLESGGGLVQPG FYAMQWVRQAPGKGLEWVS YISSSGGHTHY GSLRLSCAASGFTFS ADSVKG 807B-M0079-D10EVQLLESGGGLVQPG NYRME WVRQAPGKGLEWVS SIWSSGGLTKQ GSLRLSCAASGFTFS ADSVKG807B-M0079-H01 EVQLLESGGGLVQPG WYDMD WVRQAPGKGLEWVS RIWPSGGSTHYGSLRLSCAASGFTFS ADSVKG 807B-M0079-H05 EVQLLESGGGLVQPG QYMMGWVRQAPGKGLEWVS SISSGGWTAYA GSLRLSCAASGFTFS DSVKG 807B-M0080-A02EVQLLESGGGLVQPG WYNMG WVRQAPGKGLEWVS SIGPSGGHTMY GSLRLSCAASGFTFS ADSVKG807B-M0080-C04 EVQLLESGGGLVQPG WYSME WVRQAPGKGLEWVS SIVSSGGHTIYAGSLRLSCAASGFTFS DSVKG 807B-M0080-F10 EVQLLESGGGLVQPG DYVMNWVRQAPGKGLEWVS SIYPSGGLTRY GSLRLSCAASGFTFS ADSVKG 807B-M0081-C03EVQLLESGGGLVQPG WYWMG WVRQAPGKGLEWVS GISSSGGRTVY GSLRLSCAASGFTFS ADSVKG807B-M0081-C05 EVQLLESGGGLVQPG WYNMD WVRQAPGKGLEWVS SIGPSGGPTKYGSLRLSCAASGFTFS ADSVKG 807B-M0081-D08 EVQLLESGGGLVQPG EYTMLWVRQAPGKGLEWVS GIWPSGGPTFY GSLRLSCAASGFTFS ADSVKG 807B-M0081-E08EVQLLESGGGLVQPG MYNMY WVRQAPGKGLEWVS RIGSSGGMTDY GSLRLSCAASGFTFS ADSVKG807B-M0081-F12 EVQLLESGGGLVQPG WYLMH WVRQAPGKGLEWVS SIVPSGGTTVYGSLRLSCAASGFTFS ADSVKG 807B-M0081-G04 EVQLLESGGGLVQPG WYSMVWVRQAPGKGLEWVS VISSSGGFTGY GSLRLSCAASGFTFS ADSVKG 807B-M0081-G11EVQLLESGGGLVQPG WYDMV WVRQAPGKGLEWVS GIWPSGGFTNY GSLRLSCAASGFTFS ADSVKG807B-M0081-H03 EVQLLESGGGLVQPG WYSMV WVRQAPGKGLEWVS SIGPSGGMTRYGSLRLSCAASGFTFS ADSVKG 807B-M0081-H07 EVQLLESGGGLVQPG FYTMVWVRQAPGKGLEWVS VISPSGGLTHY GSLRLSCAASGFTFS ADSVKG 807B-M0082-B07EVQLLESGGGLVQPG YYSMT WVRQAPGKGLEWVS SISPSGGGTGY GSLRLSCAASGFTFS ADSVKG807B-M0082-E01 EVQLLESGGGLVQPG WYGMS WVRQAPGKGLEWVS WISPSGGMTKYGSLRLSCAASGFTFS ADSVKG 807B-M0082-E08 EVQLLESGGGLVQPG WYDMDWVRQAPGKGLEWVS SISSSGGFTTYA GSLRLSCAASGFTFS DSVKG 807B-M0082-H06EVQLLESGGGLVQPG WYGMG WVRQAPGKGLEWVS YISSSGGLTIYA GSLRLSCAASGFTFS DSVKG807B-M0083-B10 EVQLLESGGGLVQPG YYAMG WVRQAPGKGLEWVS WISPSGGATHYGSLRLSCAASGFTFS ADSVKG 807B-M0083-E10 EVQLLESGGGLVQPG YYSMVWVRQAPGKGLEWVS WISSSGGSTNY GSLRLSCAASGFTFS ADSVKG 807B-M0083-E11EVQLLESGGGLVQPG VYSMA WVRQAPGKGLEWVS GIWPSGGPTAY GSLRLSCAASGFTFS ADSVKG807B-M0084-C03 EVQLLESGGGLVQPG WYDMM WVRQAPGKGLEWVS YIYSSGGSTRYGSLRLSCAASGFTFS ADSVKG 807B-M0084-C11 EVQLLESGGGLVQPG FYRMAWVRQAPGKGLEWVS VISPSGGHTYY GSLRLSCAASGFTFS ADSVKG 807B-M0084-E07EVQLLESGGGLVQPG WYRMA WVRQAPGKGLEWVS SISSSGGDTQY GSLRLSCAASGFTFS ADSVKG807B-M0084-F03 EVQLLESGGGLVQPG WYVMD WVRQAPGKGLEWVS SISPSGGGTLYGSLRLSCAASGFTFS ADSVKG 807B-M0084-F08 EVQLLESGGGLVQPG WYGMAWVRQAPGKGLEWVS YISPSGGGTSY GSLRLSCAASGFTFS ADSVKG 807B-M0084-H05EVQLLESGGGLVQPG WYTME WVRQAPGKGLEWVS GIYSSGGTTTY GSLRLSCAASGFTFS ADSVKG807B-M0085-B12 EVQLLESGGGLVQPG WYGMV WVRQAPGKGLEWVS SISPSGGQTFYGSLRLSCAASGFTFS ADSVKG 807B-M0085-C01 EVQLLESGGGLVQPG WYTMDWVRQAPGKGLEWVS GISPSGGYTTY GSLRLSCAASGFTFS ADSVKG 807B-M0085-E10EVQLLESGGGLVQPG WYQMS WVRQAPGKGLEWVS GISSSGGSTQY GSLRLSCAASGFTFS ADSVKG807B-M0085-G03 EVQLLESGGGLVQPG WYVMM WVRQAPGKGLEWVS SIVPSGGGTGYGSLRLSCAASGFTFS ADSVKG 807B-M0085-G07 EVQLLESGGGLVQPG WYAMDWVRQAPGKGLEWVS SIVPSGGRTLY GSLRLSCAASGFTFS ADSVKG 807B-M0085-G08EVQLLESGGGLVQPG FYGMG WVRQAPGKGLEWVS RIRPSGGMTSY GSLRLSCAASGFTFS ADSVKG807B-M0086-C06 EVQLLESGGGLVQPG WYSMV WVRQAPGKGLEWVS WISSSGGFTKYGSLRLSCAASGFTFS ADSVKG 807B-M0086-D03 EVQLLESGGGLVQPG WYTMGWVRQAPGKGLEWVS SIVSSGGYTPY GSLRLSCAASGFTFS ADSVKG 807B-M0086-E08EVQLLESGGGLVQPG WYDMH WVRQAPGKGLEWVS WIVPSGGITEYA GSLRLSCAASGFTFS DSVKG807B-M0086-G03 EVQLLESGGGLVQPG EYKMN WVRQAPGKGLEWVS YIYPSGGFTHYGSLRLSCAASGFTFS ADSVKG Isolate Name FR3 CDR3 FR4 807B-M0011-C07RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR807B-M0012-C09 RFTISRDNSKNTLYLQMNSL GLRYFDFYYYYGMDV WGQGTTVTVSSRAEDTAVYYCAR 807B-M0012-D09 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSSRAEDTAVYYCAR 807B-M0012-F10 RFTISRDNSKNTLYLQMNSL ERGTIFNDAFDIWGQGTMVTVSS RAEDTAVYYCAR 807B-M0012-F12 RFTISRDNSKNTLYLQMNSLEPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR 807B-M0012-G05RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0013-A12RFTISRDNSKNTLYLQMNSL GSLSSGWDY WGQGTLVTVSS RAEDTAVYYCAK 807B-M0013-B04RFTISRDNSKNTLYLQMNSL EDFWSGLEDV WGKGTTVTVSS RAEDTAVYYCAR 807B-M0013-C03RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0013-F06RFTISRDNSKNTLYLQMNSL WGVRGVIPFDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0013-G05RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR807B-M0014-D07 RFTISRDNSKNTLYLQMNSL GRWLAPFDY WGQGTLVTVSS RAEDTAVYYCAR807B-M0014-D09 RFTISRDNSKNTLYLQMNSL ATCTGGSCYRFDY WGQGTLVTVSSRAEDTAVYYCAR 807B-M0014-E08 RFTISRDNSKNTLYLQMNSL GLGMDV WGQGTTVTVSSRAEDTAVYYCAR 807B-M0014-F07 RFTISRDNSKNTLYLQMNSL QEVWQWPAQFDSWGQGTLVTVSS RAEDTAVYYCAR 807B-M0016-C06 RFTISRDNSKNTLYLQMNSL DLWFGEWDYWGQGTLVTVSS RAEDTAVYYCAR 807B-M0016-D01 RFTISRDNSKNTLYLQMNSL DLWFGEWDYWGQGTLVTVSS RAEDTAVYYCAR 807B-M0016-D08 RFTISRDNSKNTLYLQMNSL DLWFGEWDYWGQGTLVTVSS RAEDTAVYYCAR 807B-M0016-E01 RFTISRDNSKNTLYLQMNSLEPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR 807B-M0016-F04RFTISRDNSKNTLYLQMNSL DFFTSYFDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0016-F05RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0016-F08RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0017-B05RFTISRDNSKNTLYLQMNSL EGVIAVAGPYRD WGQGTLVTVSS RAEDTAVYYCAK807B-M0017-B06 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR807B-M0017-E05 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR807B-M0018-C12 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR807B-M0018-E09 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR807B-M0018-G02 RFTISRDNSKNTLYLQMNSL GERAAAGTQHYYYY WGQGTTVTVSSRAEDTAVYYCAR YGMDV 807B-M0019-A04 RFTISRDNSKNTLYLQMNSL GRSLYYDFWSGYYPWGKGTTVTVSS RAEDTAVYYCAR NTYYYYYMDV 807B-M0019-A10 RFTISRDNSKNTLYLQMNSLGGGFGVYHHYYDM WGQGTTVTVSS RAEDTAVYYCAR DV 807B-M0019-C01RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0019-F06RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0019-G07RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0020-D01RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0020-F06RFTISRDNSKNTLYLQMNSL DTSGWYEEEDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0020-F12RFTISRDNSKNTLYLQMNSL DLWFGEWDH WGQGTLVTVSS RAEDTAVYYCAR 807B-M0020-G01RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0079-B09RFTISRDNSKNTLYLQMNSL GAGALGY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0079-D10RFTISRDNSKNTLYLQMNSL GLYR WGQGTLVTVSS RAEDTAVYYCAR 807B-M0079-H01RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR807B-M0079-H05 RFTISRDNSKNTLYLQMNSL DGGTWDFDY WGQGTLVTVSS RAEDTAVYYCAK807B-M0080-A02 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR807B-M0080-C04 RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDV WGQGTTVTVSSRAEDTAVYYCAR 807B-M0080-F10 RFTISRDNSKNTLYLQMNSL GAGALGY WGQGTLVTVSSRAEDTAVYYCAR 807B-M0081-C03 RFTISRDNSKNTLYLQMNSL GHWGWFDP WGQGTLVTVSSRAEDTAVYYCAR 807B-M0081-C05 RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDVWGQGTTVTVSS RAEDTAVYYCAR 807B-M0081-D08 RFTISRDNSKNTLYLQMNSLELDTAMAPPSDAFDI WGQGTMVTVSS RAEDTAVYYCAR 807B-M0081-E08RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR807B-M0081-F12 RFTISRDNSKNTLYLQMNSL DLWFGEWDY RAEDTAVYYCAR807B-M0081-G04 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR807B-M0081-G11 RFTISRDNSKNTLYLQMNSL HPVSSGFDY WGQGTLVTVSS RAEDTAVYYCAR807B-M0081-H03 RFTISRDNSKNTLYLQMNSL DQGITMVQGAMGY WGQGTLVTVSSRAEDTAVYYCAR 807B-M0081-H07 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSSRAEDTAVYYCAR 807B-M0082-B07 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSSRAEDTAVYYCAR 807B-M0082-E01 RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSSRAEDTAVYYCAR 807B-M0082-E08 RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDVWGQGTTVTVSS RAEDTAVYYCAR 807B-M0082-H06 RFTISRDNSKNTLYLQMNSL DLWFGEWDYWGQGTLVTVSS RAEDTAVYYCAR 807B-M0083-B10 RFTISRDNSKNTLYLQMNSL DLWFGEWDYWGQGTLVTVSS RAEDTAVYYCAR 807B-M0083-E10 RFTISRDNSKNTLYLQMNSL DLWFGEWDYWGQGTLVTVSS RAEDTAVYYCAR 807B-M0083-E11 RFTISRDNSKNTLYLQMNSL EDFWSGLEDVWGKGTTVTVSS RAEDTAVYYCAR 807B-M0084-C03 RFTISRDNSKKTLYLQMNSLEPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR 807B-M0084-C11RFTISRDNSKNTLYLQMNSL DFFTSYFDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0084-E07RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0084-F03RFTISRDNSKNTLYLQMNSL DLHYGSVLDF WGQGTLVTVSS RAEDTAVYYCAR 807B-M0084-F08RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0084-H05RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR807B-M0085-B12 RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDV WGQGTTVTVSSRAEDTAVYYCAR 807B-M0085-C01 RFTISRDNSKNTLYLQMNSL GTVLLWFGESGGHFWGQGTPVTVSS RAEDTAVYYCAR DY 807B-M0085-E10 RFTISRDNSKNTLYLQMNSLDLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0085-G03 RFTISRDNSKNTLYLQMNSLENYGPDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0085-G07 RFTISRDNSKNTLYLQMNSLEPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR 807B-M0085-G08RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0086-C06RFTISRDNSKNTLYLQMNSL DLWFGEWDY WGQGTLVTVSS RAEDTAVYYCAR 807B-M0086-D03RFTISRDNSKNTLYLQMNSL EPIWGYYYYGMDV WGQGTTVTVSS RAEDTAVYYCAR807B-M0086-E08 RFTISRDNSKNTLYLQMNSL QEVWQWPAQFDS WGQGTLVTVSSRAEDTAVYYCAR 807B-M0086-G03 RFTISRDNSKNTLYLQMNSL SVVGWGLDY WGQGTLVTVSSRAEDTAVYYCAR

TABLE 16 Enrichment Peptide mapping koff Fab Isolate Name VH CDR3 bCTDVLDL 1 2 3 4 5 6 7 8 9 10 (e−3) RU IHC 807B-M0011-C07 3 20 + 11.6 325807B-M0012-C09 12 12 + 11.7 156 807B-M0012-D09 1 299 + 14.8 159807B-M0012-F10 1 1 + 5.5 50.5 807B-M0012-F12 2 20 + 3.0 246807B-M0012-G05 15 299 + 7.9 157 807B-M0013-A12 3 3 + 6.8 63.9 +807B-M0013-B04 1 4 + 12.5 101 807B-M0013-C03 1 299 + + 38.4 177807B-M0013-F06 1 1 + + + 9.3 162 807B-M0013-G05 1 20 + +/− + 17.5 136807B-M0014-D07 10 10 + + 8.1 206 807B-M0014-D09 13 13 + + 5.2 80.4807B-M0014-E08 2 2 + + 18.6 151 807B-M0014-F07 1 2 + 8.6 205807B-M0016-C06 5 299 + 7.2 74.2 807B-M0016-D01 1 299 + 8.5 74807B-M0016-D08 1 299 + + 12.4 80.3 807B-M0016-E01 1 20 + 8.0 465807B-M0016-F04 1 2 + 13.5 58.9 807B-M0016-F05 15 299 + 9.0 69807B-M0016-F08 1 299 + 18.5 113 807B-M0017-B05 1 1 + +/− 26.1 248807B-M0017-B06 1 299 + 8.8 120 807B-M0017-E05 1 299 + 4.7 71.1807B-M0018-C12 2 299 + 11.0 107 807B-M0018-E09 3 299 + 10.5 66.8807B-M0018-G02 1 1 + + 21.1 98.9 807B-M0019-A04 1 1 + 5.4 31.1807B-M0019-A10 1 1 + 7.1 51 807B-M0019-C01 1 299 + 10.6 80.1807B-M0019-F06 1 299 + 7.7 99.4 807B-M0019-G07 1 299 + 5.2 31.2807B-M0020-D01 1 299 + 3.9 51.2 807B-M0020-F06 1 1 + 4.9 53.2807B-M0020-F12 1 1 + 9.9 67.4 807B-M0020-G01 1 299 + 12.7 68807B-M0079-B09 1 2 + +/+ + 8.3 438 807B-M0079-D10 + + + + 4.3 100 +807B-M0079-H01 1 20 + + + + 2.5 256 807B-M0079-H05 1 1 + 2.0 99.3807B-M0080-A02 1 299 + 2.5 86.8 807B-M0080-C04 1 20 + 2.5 162807B-M0080-D06 1 1 + + 2.4 72.7 807B-M0080-F10 1 2 + + 2.7 49.2807B-M0081-C03 1 1 + + 2.2 84.6 807B-M0081-C05 1 20 + 2.6 158807B-M0081-D08 1 1 + + 4.0 55.1 807B-M0081-E08 1 20 + 5.7 65.1807B-M0081-F12 249 299 + 6.1 128 + 807B-M0081-G04 1 299 + 2.6 64.1807B-M0081-G11 1 1 + + 3.6 83.6 807B-M0081-H03 1 1 + 9.7 346 +/−807B-M0081-H07 1 299 + 2.3 56.4 807B-M0082-B07 1 299 + +/− 2.8 46.4807B-M0082-C11 1 1 + 4.0 244 807B-M0082-E01 2 299 + + + 4.7 68.8807B-M0082-E08 1 20 + 3.0 154 807B-M0082-F04 1 20 + 3.1 136807B-M0082-F11 1 299 + 6.1 32.5 807B-M0082-H06 2 299 + + 5.1 103807B-M0083-B10 1 299 + + + 2.9 45 807B-M0083-E10 1 299 + + 3.7 47.9807B-M0083-E11 3 4 + 5.1 34.6 + 807B-M0084-C03 1 20 + 2.0 53.9807B-M0084-C11 1 2 + 3.7 52.5 807B-M0084-E07 1 299 + + + + 4.4 46.7807B-M0084-F03 1 1 + 3.6 49.6 807B-M0084-F08 3 299 + 5.8 51.5807B-M0084-H05 2 20 + + 2.4 118 807B-M0085-B12 1 20 + + 7.9 95.3807B-M0085-C01 1 1 + 5.5 69.7 807B-M0085-E01 1 20 + 2.8 49.5807B-M0085-E10 1 299 + + + + + + + + 5.8 47.5 807B-M0085-G03 1 1 + 2.252.8 807B-M0085-G07 1 20 + 4.2 83.7 807B-M0085-G08 1 299 + + 3.8 42.2807B-M0086-C06 1 299 + 4.8 59.5 807B-M0086-D03 1 20 + 3.7 159807B-M0086-E08 1 2 + 2.6 73 807B-M0086-G03 2 2 + 3.7 43.8

TABLE 17 Fab Binding Data to CTD and peptides CTD Affinity Biacore (nM)VLDL Isolate name Strategy IHC ELISA peptide group CTD p1 p4 p6 p8 p9ELISA 807B-M0001-B07 E22 P1 +/− + p1, p6 4 46 245 396 − 807B-M0004-A03E22 P4 ++ + p4, p9 5 low 98 low − RU* RU* 807B-M0004-A05 E22 P4 +/− − p41 no 208 − RU** 807B-M0004-C04 E22 P4 +/− − p4 1 no 509 − RU**807B-M0004-C05 E22 P4 +/− − p4 1 no 225 − RU** 807B-M0004-F06 E22 P4 +/−− p4 1 no 361 − RU** 807B-M0004-F10 E22 P4 +/− − p4, p9 2 no 104 low −RU** RU* 807B-M0004-H03 E22 P4 ++ − p4, p9 2 no 200 low − RU** RU*807B-M0009-C03 E22 P9, +/− + p4, p9 5 no 728 163 − E24 P3 RU**807B-M0009-F06 E22 P9, +/− − p9 3 no 172 − E24 P3 RU** 807B-M0013-A12E24 ++ + no 7 no − RU** 807B-M0079-D10 E22&E23 ++ + p4, p8 6 59.17 low25.79 − P9, E24 RU* 807B-M0081-F12 E24 + + no 8 low − RU* 807B-M0081-H03E24 +/− + no 9 10 − 807B-M0083-E11 E24 ++ + no 10 low − RU*807A-M0028-B02 E5 + + no − 807A-M0026-F05 E5 + + p3, p8 − 807A-M0027-E11E5 + + no − *low RU: RUs are low, affinity was not measured **no RU: nobinding observed

TABLE 18 IgG Binding Data to CTD and peptides CTD Affinity Biacore (nM)Isolate name Strategy IHC ELISA peptide hCTD mCTD pCTD 807B-M0001-B07E22 P1 + + p1, p6 5 <h, pCTD 6 807B-M0004-A03 E22 P4 +++ + (p1, p6) 3 3p4, p8, p9 807B-M0004-A05 E22 P4 − + p4, p9 167 no RU** no RU**807B-M0004-C04 E22 P4 − +/− p4, p9 no RU** no RU** no RU**807B-M0004-C05 E22 P4 − − p4, p9 no RU** no RU** no RU** 807B-M0004-F06E22 P4 − no RU** no RU** no RU** 807B-M0004-F10 E22 P4 (+) + p4, p8, p925 no RU** 23 807B-M0004-H03 E22 P4 +(+) + p4, p9, 45 no RU** 43 (p8)807B-M0009-C03 E22 P9, (+) + p4, p9 23 no RU** 4 E24 P3 807B-M0009-F06E22 P9, ++ − p4, p9 34 234 28 E24 P3 807B-M0013-A12 E24 +++ + (p3, p9),27 23 26 p4, p8 807B-M0079-D10 E22&E23 (+) + p4, p8 1 2 1 P9, E24807B-M0081-F12 E24 + (p9) 15 16 19 807B-M0081-H03 E24 (+) + (p7) 9 18 7807B-M0083-E11 E24 − + no 12 64 25 807A-M0028-B02 E5 + + P4 9 7 5807A-M0026-F05 E5 + 807A-M0027-E11 E5 + VLDL Affinity Biacore (nM) ELISAELISA Isolate name p1 p4 p8 p9 (O.D)† hCTD mCTD pCTD 807B-M0001-B07 8.042.14 +++ ++ +++ 807B-M0004-A03 0.17 low RU* 0.273 + + ++ 807B-M0004-A0517.42 0.214 +/− +/− +/− 807B-M0004-C04 8.68 0.242 +/− +/− +/−807B-M0004-C05 0.97 0.233 +/− +/− +/− 807B-M0004-F06 6.72 0.248 +/− +/−+/− 807B-M0004-F10 1.00 13.74 1.11 ++ +/− +/− 807B-M0004-H03 11.55 lowRU* 0.309 ++ +(+) ++ 807B-M0009-C03 1.00 0.27 2.528 +++ +++ +++807B-M0009-F06 no RU** 10.6 1.202 +/− +/− +/− 807B-M0013-A12 0.462 +++/− +/− 807B-M0079-D10 43.64 1.05 0.2 +++ +++ +++ 807B-M0081-F12 no RU**0.285 ++ + + 807B-M0081-H03 no RU** 2.88 +++ +++ +++ 807B-M0083-E11 noRU** 0.221 + − − 807A-M0028-B02 2.679 807A-M0026-F05 − 807A-M0027-E11 −*low RU: RUs are low, affinity was not measured **no RU: no bindingobserved †VLDL ELISA: VLDL is coated, 5 μg/ml of appropriate hIgG isadded O.D. of 2x background is 0.420

TABLE 19 Amino acid sequences of the VL chains of the Germline-correctedantibodies Initial Name LV-FR1 LV-CDR1 LV-FR2 LV-CDR2 807A-M0028-B02.1DIQMTQSPSSLSA RTSQDIRNHLG WFQQKPGKAP EASILQS SVGDRVTITC QRLIR807A-M0028-B02.2 DIQMTQSPSSLSA RTSQDIRNHLG WYQQKPGKAP EASILQS SVGDRVTITCKRLIY 807B-M0004-H03.1 DIQMTQSPSSLSA QASQNIDNYLN WYQQKPGKAP AASSLQSSVGDRVTITC KLLIY 807B-M0009-F06.1 DIVMTQSPLSLPV KSSQSLLHSNGYNYLDWYLQKPGQSP LGSNRAS TPGEPASISC QLLIY 807B-M0004-A03.1 QSVLTQPPSASGSGSSSNIGSNTVN WYQQLPGTAP NNNQRPS TPGQRVTISC KLLIY 807B-M0079-D10.1SYELTQPPSVSVS AGDELGNKYAS WYQQKPGQSP QDRKRPS PGQTASITC VLVIY InitialName LV-FR3 LV-CDR3 LV-FR4 807A-M0028-B02.1 GVPSTFYGSGYGREFTLTISLQYDSFPYT FGQGTKLEIK SLQPEDFATYYC 807A-M0028-B02.2 GVPSRFSGSGSGTEFTLTISLQYDSFPYT FGQGTKLEIK SLQPEDFATYYC 807B-M0004-H03.1 GVPSRFSGSGSGTDFTLTISQQSYSTPRT FGQGTKVEIK SLQPEDFATYYC 807B-M0009-F06.1 GVPDRFSGSGSGTDFTLKISMQALQTIT FGQGTRLEIK RVEAEDVGVYYC 807B-M0004-A03.1 GVPDRFSGSKSGTSASLAISAAWHDGLNGPV FGGGTKLTVL GLQSEDEADYYC 807B-M0079-D10.1GIPERFSGSNSGNTATLTIS QSWDSSSVI FGGGTKLTVL GTQAMDEADYYC

TABLE 20 Amino acid sequences of the CL chains of the Germline-correctedantibodies Name Germ line Sequence of constant region of light chain807A-M0028-B02.1 KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 807A-M0028-B02.2 KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 807B-M0004-A03.1 LambdaGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS 807B-M0079-D10.1 LambdaGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

TABLE 21 Description of SEQ ID NOS: 539-548 Antibody VL chain sequenceCL chain sequence 807A-M0028-B02.1 SEQ ID NO: 518 SEQ ID NO: 519807A-M0028-B02.2 SEQ ID NO: 520 SEQ ID NO: 521 807B-M0004-H03.1 SEQ IDNO: 524 — 807B-M0009-F06.1 SEQ ID NO: 525 — 807B-M0004-A03.1 SEQ ID NO:522 SEQ ID NO: 523 807B-M0079-D10.1 SEQ ID NO: 526 SEQ ID NO: 527

TABLE 22 IgG Binding Data to CTD and peptides Isolate CTD AffinityBiacore (nM) VLDL ELISA⁶ name Strategy IHC² ELISA peptide hCTD mCTD pCTDp1 p4 p8 p9 ELISA⁵ hCTD mCTD pCTD 807B- E22 P1 + + p1, p6 5 <h, pCTD 68.04 ++ +++ ++ +++ M0001- B07 807B- E22 P4 +++ + (p1, p6) 3 3 0.17 low++ +++ ++ + M0004- p4, p8, RU⁴ A03 P9 807B- E22 P4 − + p4, p9 167 no RU³no RU³ 17.42 − +/− +/− +/− M0004- A05 807B- E22 P4 − +/− p4, p9 no RU³no RU³ no RU³ 8.68 − +/− +/− +/− M0004- C04 807B- E22 P4 − − p4, p9 noRU³ no RU³ no RU³ 0.97 − +/− +/− +/− M0004- C05 807B- E22 P4 − no RU³ noRU³ no RU³ 6.72 − +/− +/− +/− M0004- F06 807B- E22 P4 (+) + p4, p8, 25no RU³ 23 1.00 13.74 + ++ +/− +/− M0004- p9 F10 807B- E22 P4 +(+) + p4,p9, 45 no RU³ 43 11.55 low ++ + − − M0004- (p8) RU⁴ H03 807B- E22 P9,(+) + p4, p9 23 no RU³ 4 1.00 0.27 ++ +++ +++ +++ M0009- E24 P3 C03807B- E22 P9, ++ − p4, p9 34 234 28 no RU³ 10.6 +++ +++ + + M0009- E24P3 F06 807B- E24 +++ + (p3, p9), 27 23 26 ++ ++ +/− +/− M0013- p4, p8A12 807B- E22&E23 (+) + p4, p8 1 2 1 43.64 1.05 +++ +++ +++ +++ M0079-P9, E24 D10 807B- E24 ¹n.d + (p9) 15 16 19 no RU³ + ++ + + M0081- F12807B- E24 (+) + (p7) 9 18 7 no RU³ +++ +++ +++ +++ M0081- H03 807B- E24− + no 12 64 25 no RU³ − + − − M0083- E11 807A- E5 + + P4 9 7 5 +++ ++++++ +++ M0028- B02 807A- E5 (+) +++ +++ ¹n.d ¹n.d M0026- F05 807A- E5 ++++ +++ ¹n.d ¹n.d M0027- E11 ¹n.d.: Not done ²IHC: tissue from human ADcortex used. 1 μg/ml of appropriate hIgG added ³no RU: no bindingobserved ⁴low RU: RUs are low, affinity was not measured ⁵VLDL ELISA:human VLDL coated 0.0003-100 μg/ml of appropriate hIgG tested forbinding ⁶ELISA: human, mouse or primate CTD coated, 0.0003-100 μg/ml ofappropriate hIgG tested for binding

TABLE 23 Binding of germ line corrected IgG to CTD and tissue Affinity(Biacore VLDL IHC³ ELISA⁴ In vitro phagocytosis Isolate name Peptide(nM)) ELISA² (tissue) hCTD mCTD pCTD EC50 ± SEM (ng/ml) 807A-M0028-B02P4 10.8 +++ ++ +++ +++ +++ 34 +/− 15 807A-M0028-B02.1 n.d.¹ 13 ++ ++ ++++++ ++ 67 +/− 38 807A-M0028-B02.2 n.d.¹ +++ ++ + + + 27 +/− 7 807B-M0004-H03 p4, p9, (p8) 12.5 ++ ++ + − + 53 +/− 25 807B-M0004-H03.1n.d.¹ 17.7 − +++ +++ − ++ 22 +/− 16 807B-M0009-F06 p4, p9 14.8 +++ +++++ + ++ 23 +/− 9  807B-M0009-F06.1 n.d.¹ very − + − − − 42 +/− 17 lowbinding 807B-M0004-A03 (p1, p6) p4, 12.2 ++ ++ +++ + ++ 205 +/− 81  p8,p9 807B-M0004-A03.1 n.d.¹ 24.4 − +³* +++ + + 78 +/− 43 807B-M0079-D10p4, p8 n.d.¹ +++ ++ +++ +++ +++   7 +/− 1.4 807B-M0079-D10.1 n.d.¹ n.d.¹++ ++ +++ +++ +++ 19 +/− 15 ¹n.d.: Not done. ²VLDL ELISA: human VLDL iscoated 0.0003-100 μg/ml of appropriate hIgG is tested for binding ³IHC:Brain tissue sections from APP/PS1 mouse was used. 1.5-1.8 μg/ml ofappropriate hIgG is added, ³*reactivity to mCTD lower than to hCTD ⁴CTDELISA: human, mouse or primate CTD is coated, 0.0003-100 μg/ml ofappropriate hIgG is tested for binding

TABLE 24 807B-M0004-A03 = 39 clones selected for Fab production AminoAc1(WT = S) 2(WT = I) 3(WT = A) 4(WT = A) 5(WT = A) 6(WT = G) 7(WT = T)8(WT = D) 9(WT = Y) A 88% 65% 60% D 9% 98% 2% E 7% 2% G 2% 5% 2% 84% H2% 21% I 98% 2% K 5% L 2% M 2% N 5% 10% P 5% Q 2% R 5% 16% 2% 5% S 91%5% 14% 19% T 2% 2% 7% 79% V 5% Y 70%

TABLE 25 807B-M0004-H03 = 54 clones selected for Fab production: part 11(WT = AminoAc E) 2(WT = G) 3(WT = S) 4(WT = A) 5(WT = G) 6(WT = V) 7(WT= V) 8(WT = K) 9(WT = G) 10(WT = P) A 2% 2% 5% 82% 9% 5% 4% 5% D 4% 2% E95% 2% 4% F 4% 4% 2% G 73% 2% 46% 73% H 2% I 7% 7% K 2% 84% L 5% 9% 9%5% M 2% 4% 2% N 4% P 5% 2% 77% Q 2% 2% R 18% 2% 9% 38% 4% 2% 11% 5% S 4%75% 4% T 2% V 4% 7% 4% 66% 77% W 4% 9% Y Stop 2%

TABLE 26 807B-M0004-H03 = 54 clones selected for Fab production: part 211(WT = 12(WT = AminoAc A) R) 13(WT = Y) 14(WT = Y) 15(WT = Y) 16(WT =Y) 17(WT = Y) 18(WT = M) 19(WT = D) 20(WT = V) A 73% 4% 4% C 2% D 2% 2%77% E 2% 4% F 2% 4% 5% 5% G 5% 2% 2% 2% H 7% 7% 2% 4% I 5% 7% K 4% L 2%2% 2% 9% M 93% N 2% 4% 4% 4% 5% P 4% 2% 2% Q 4% 5% R 2% 83% S 5% 4% 5%2% T 5% V 4% 2% 71% Y 4% 88% 92% 85% 93% 82% 2%

TABLE 27 807B-M0079-D10 = 33 clones selected for Fab production AminoAc1(WT = G) 2(WT = L) 3(WT = Y) 4(WT = R) A 11% 6% D 3% F 9% G 69% 3% H11% 9% I 3% K 3% L 86% 3% 17% M 6% N 6% P 3% Q 3% 9% R 43% S 14% 6% 3% T3% V 3% 3% W 3% Y 57% 6% Stop 2%

TABLE 28 807A-M0028-B02-CTD = 60 clones selected for Fab productionAminoAc 1(WT = S) 2(WT = V) 3(WT = L) 4(WT = L) 5(WT = D) 6(WT = Y) A18% 2% 2% 2% D 2% 92% E 2% 2% F 2% 19% 3% G 19% 3% H 3% 23% 2% 11% I23%  2% 2% 2% K 16% L 2% 87% 52% 2% M 3% N 2% 11% P 2% 2% 3% 3% Q 2% 3%R 2% 3% S 57% 8% T 2% 2% 2% 3% V 66.50%    Y 2% 2% 29%

TABLE 29 807A-M0028-B02-fibrils = 12 clones selected for Fab production1(WT = 2(WT = 3(WT = 5(WT = 6(WT = AminoAc S) V) L) 4(WT = L) D) Y) A33% D 100% F 25% G H 8% 17% 17% I K 8% L 92% 58% N 17% Q 8% R 8% S 67%17% V 92% Y 33%

TABLE 30 807B-M0009-F06 = 24 clones selected for Fab production 1(WT =AminoAc V) 2(WT = G) 3(WT = M) 4(WT = S) 5(WT = T) 6(WT = Y) 7(WT = A)8(WT = F) 9(WT = D) 10(WT = I) A 8% 8% 24% D 96% E 4% F 4% 96% 4% G 92%60% H 4% I 20% 56% K 4% 14% L 12% 4% 4% 16% M 60% 4% N 12% P 8% S 60% 8%16% T 12% 76% 8% V 100% 4% 8% Y 4% 100%

TABLE 31 Biacore analysis of 807B-M0004-A03, original clone and variantsBC koff kon KD Ranking Clone HV-CDR3 nM 1/s 1/Ms nM koff KD *807B-M0004-SIAAAGTDY 234 0.0294 3.18E+05 9.26E−08 27 13 A03/WT 807B-M0004-SIAAAGTDY 232 0.0275 2.54E+05 1.08E−07 23 16 A03/WT 807B-M0004-SIAADGIDY 181 0.0341 ND 3.07E+03 33 41 A03/M0117-A04 807B-M0004-SIAATRTDY 239 0.0215 1.47E+05 1.46E−07 10 28 A03/M0117-A12 807B-M0004-SIAAARTEY 192 0.0262 3.29E+04 7.97E−07 21 36 A03/M0117-B03 807B-M0004-SIAPSGTDY 210 0.132 6.22E−04 212     43 40 A03/M0117-B04 807B-M0004-SIAPAGTDH 224 0.0297 2.45E+05 1.21E−07 28 22 A03/M0117-B05 807B-M0004-SIAEAGTDY 245 0.0464 3.02E+05 1.54E−07 35 29 A03/M0117-B11 807B-M0004-SIAVAGTDY 193 0.0902 1.25  0.072 41 38 A03/M0117-C04 807B-M0004-SIAGAGNDY 186 0.0306 2.40E+05 1.28E−07 30 23 A03/M0117-C07 807B-M0004-SIAAAGTDH 280 0.0188 2.53E+05 7.44E−08 6 10 A03/M0117-C09 807B-M0004-SIGAAGTDY 263 0.0767 0.0967 0.788 39 39 A03/M0117-C11 807B-M0004-SIAASGTDY 134 0.0284 3.95E+05 7.22E−08 26 8 A03/M0117-C12 *807B-M0004-SIAAARTDY 199 0.0128 9.45E+04 1.34E−07 3 25 A03/M0117-D03 807B-M0004-SIQAAGTDH 123 0.0247 1.96E−05 4.21E+03 15 42 A03/M0117-E06 807B-M0004-SIASPGTDY 228 0.0266 1.95E+05 1.37E−07 22 27 A03/M0117-E12 *807B-M0004-SIASAGTDH 290 0.0139 2.22E+05 6.13E−08 4 4 A03/M0117-F05 807B-M0004-GISTSGTDD 284 0.0243 2.08E+05 1.15E−07 14 19 A03/M0117-F11 807B-M0004-SIAVAGTDY 235 0.0906 1.82E−05 4.96E+03 42 43 A03/M0117-F12 *807B-M0004-SIASARTDS 256 0.0103 1.66E+05 6.07E−08 1 3 A03/M0117-G01 807B-M0004-SIAAPGTDY 220 0.011 1.92E+04 4.84E−07 2 35 A03/M0117-G03 807B-M0004-RIAASGTDY 92 0.049 4.42E+05 1.20E−07 36 21 A03/M0117-G04 807B-M0004-SIAATGKDH 280 0.0815 9.94E+04 8.24E−07 40 37 A03/M0117-G05 807B-M0004-SIAAAGSDS 471 0.0283 1.35E+05 2.23E−07 25 33 A03/M0117-G07 807B-M0004-SIGASRTDY 336 0.0502 3.42E+05 1.68E−07 37 31 A03/M0117-H06 807B-M0004-SIASAGTDL 217 0.0205 2.87E+05 6.97E−08 8 6 A03/M0117-H11 807B-M0004-SIAAAGNDY 167 0.0254 3.51E+05 7.12E−08 17 7 A03/M0118-A03 *807B-M0004-SIAADRTDY 252 0.0231 4.78E+05 4.70E−08 11 1 A03/M0118-B09 807B-M0004-SIAESGTDY 203 0.0298 3.34E+05 9.06E−08 29 12 A03/M0118-B11 807B-M0004-SIASSGTDH 213 0.0205 3.64E+05 5.47E−08 9 2 A03/M0118-C04 807B-M0004-RMAAAGTDY 225 0.0235 9.81E+04 2.42E−07 12 34 A03/M0118-D02 807B-M0004-SIAAAGKDY 281 0.0306 2.90E+05 1.33E−07 31 24 A03/M0118-D03 807B-M0004-SIAATGTDI 263 0.028 2.54E+05 1.19E−07 24 20 A03/M0118-D07 807B-M0004-SIAAAGNDH 179 0.0236 3.19E+05 7.41E−08 13 9 A03/M0118-E10 807B-M0004-SIASAGTDY 267 0.0312 2.52E+05 1.55E−07 32 30 A03/M0118-E12 *807B-M0004-SIAASRTDY 230 0.0158 2.36E+05 6.56E−08 5 5 A03/M0118-F03 807B-M0004-SIAAAGTDH 372 0.0194 2.38E+05 7.97E−08 7 11 A03/M0118-F06 807B-M0004-SIAEAGTDY 245 0.0456 2.92E+05 1.78E−07 34 32 A03/M0118-F09 807B-M0004-SISAAGTDY 278 0.0249 1.78E+05 1.37E−07 16 26 A03/M0118-F12 807B-M0004-SIAADGTDY 208 0.0669 6.16E+05 1.14E−07 38 18 A03/M0118-G03 807B-M0004-TIAAAGTDY 267 0.0259 2.42E+05 1.13E−07 20 17 A03/M0118-G05 807B-M0004-SIAAAGHDH 336 0.0256 2.70E+05 1.00E−07 18 14 A03/M0118-G08 807B-M0004-SIAAAGNDY 276 0.0258 2.69E+05 1.00E−07 19 15 A03/M0118-H01 *= selectedclone BC = Biacore

TABLE 32 Biacore analysis of 807B-M0004-H03, original clone and variantsBiacore koff kon KD Ranking Clone HV-CDR3 nM 1/s 1/Ms nM koff KD*807B-M0004-H03/WT EGSAGVVKGPARYYYYYMDV 703 1.18E−03 4.37E+03 2.56E−07 116 807B-M0004-H03/M0119-A07 ERSAGVLKGPAWYYYYYMDV 262 5.94E−03 1.48E+041.67E−07 36 7 807B-M0004-H03/M0119-A08 EGSAAFVKGPARYYYYDMDI 375 2.66E−011.24E+04 4.64E−07 55 30 *807B-M0004-H03/M0119-B05 EGSSGVVKGPARYYYYYMDA371 1.53E−03 6.03E+03 1.59E−07 4 5 807B-M0004-H03/M0119-B06EGSVGAVKGRARYYYYYMNV 729 2.62E−03 3.45E+03 4.00E−07 16 26807B-M0004-H03/M0119-B07 EGSAGVFKGPARYYYYYMDV 486 7.20E−03 1.36E+043.98E−07 40 24 *807B-M0004-H03/M0119-C05 ERSVAVFKARPRHYYYYMDV 6961.69E−03 1.09E+04 1.46E−07 5 3 807B-M0004-H03/M0119-C08EGSAGVDIGPARYYYYYMNV 453 7.24E−03 1.46E+04 4.72E−07 41 32807B-M0004-H03/M0119-C11 EGSAAVVKAPAKYYYYYMEV 347 7.39E−03 1.47E+044.70E−07 43 31 807B-M0004-H03/M0119-E01 EGSVGVVKGPARYYHYQIDV 5413.03E−03 7.69E+03 3.29E−07 19 21 807B-M0004-H03/M0119-E04ESSARVVKGLARYYNYYMHV 484 5.34E−03 5.43E+03 6.00E−07 31 38807B-M0004-H03/M0119-E07 ERPSRVVKGPTRYYYYYMDV 577 5.51E−03 4.33E−037.70E−01 32 52 807B-M0004-H03/M0119-E11 EVSARVVKCPARYYYYYMDV 6149.69E−03 7.74E−06 1.19E+03 45 54 807B-M0004-H03/M0119-F01EGSAGVIKGPARYYYFYMGV 389 1.85E−01 1.38E+04 7.14E−07 54 41*807B-M0004-H03/M0119-F04 EGSARVIKGPARYYYYEMDV 675 3.99E−03 2.68E+041.44E−07 29 2 807B-M0004-H03/M0119-F05 ERSVGVVIGHARYFYYYMDV 472 1.72E−038.05E+03 1.99E−07 6 8 807B-M0004-H03/M0119-F09 EGPAGVVKGRARYYSYNMSV 2461.13E−01 8.96E+05 1.10E−07 52 1 807B-M0004-H03/M0119-F10ESSARVVNGPAWYYYYYMDA 261 5.71E−03 8.52E+03 6.47E−07 35 39807B-M0004-H03/M0119-F11 EGSSRAVKGAPRYYYYYMDV 1027 3.41E−03 1.52E+031.68E−06 24 45 807B-M0004-H03/M0119-F12 EVSGGVVKGPARYYYYYMAL 8622.57E−02 6.16E+03 3.93E−06 48 47 807B-M0004-H03/M0119-G08EGqARRVKGQARYYYYYMDV 270 3.33E−03 3.29E+03 6.81E−07 23 40807B-M0004-H03/M0119-G10 EGSAGLVKGPARYYYYYMDV 514 1.76E−03 4.94E+032.95E−07 7 19 807B-M0004-H03/M0119-G12 ERSAGVVKGPSRNYYYYMDV 728 3.73E−031.56E+04 2.36E−07 27 14 807B-M0004-H03/M0119-H01 EGSARRVKRPGRYYYYQMDV912 1.06E−02 3.66E+04 2.84E−07 46 18 807B-M0004-H03/M0119-H03EGSARMLKGPARCYYYYMDV 315 7.36E−03 1.55E+04 4.38E−07 42 28807B-M0004-H03/M0119-H08 EGMAGVVKFPARHNYHYMDV 881 4.58E−02 8.86E+045.76E−07 49 37 *807B-M0004-H03/M0119-H09 DGSARVVKGPRRYYYYYIDV 3511.40E−03 7.92E+03 1.50E−07 2 4 807B-M0004-H03/M0119-H11ERPAGLVKGPARYYSYYMDV 1205 2.21E−03 6.55E+03 3.11E−07 12 20807B-M0004-H03/M0120-A03 EGSARMVKGAARYYYYYMDV 1109 1.62E−02 6.25E+042.50E−07 47 15 807B-M0004-H03/M0120-A07 EGSAGTIKWLVRYYNFYMDV 7073.24E−03 6.77E+03 4.48E−07 21 29 *807B-M0004-H03/M0120-B05EGSARVVKGPARYFYYYMDL 740 1.40E−03 4.70E+03 2.34E−07 3 13807B-M0004-H03/M0120-B06 EGSARVVKGPDRYYYYYMAP 309 5.62E−03 8.84E−034.82E−01 33 51 807B-M0004-H03/M0120-B09 EGSAGKVIGPAPHYYYYMDV 8686.90E−03 2.05E+04 3.34E−07 39 22 807B-M0004-H03/M0120-B11EGRARVLKGLARYYHYYMDF 1023 3.01E−03 3.50E+02 5.42E−06 18 49807B-M0004-H03/M0120-C02 EGSARFVKGPARYYYYYMDI 779 3.74E−03 5.68E+024.10E−06 28 48 807B-M0004-H03/M0120-C06 EGSSRLVQWPARYYYYSMDV 7473.67E−03 5.96E+03 5.18E−07 26 35 807B-M0004-H03/M0120-C07ERSAGVMKGPTLYYYYYMDV 641 8.85E−03 6.51E+03 1.17E−06 44 44807B-M0004-H03/M0120-C12 EGSAGVVNRSSRYNYYYLDV 959 4.94E−02 ND ND 50 —807B-M0004-H03/M0120-D04 EGSSVEVKGPARYYHYYMDV 652 1.85E−03 6.71E+032.07E−07 10 9 807B-M0004-H03/M0120-D05 EGSAGVVKGPTRYYYYSMDV 921 2.28E−033.84E+03 4.81E−07 15 33 807B-M0004-H03/M0120-E02 EGSAVVVKRSARYYYYYMNF827 6.72E−03 6.74E+03 8.20E−07 38 42 807B-M0004-H03/M0120-E04ERSARLLKGPLRYYYYYMDV 817 1.86E−03 2.35E+03 3.70E−07 11 23807B-M0004-H03/M0120-E06 ERAARAVKGPSRYYYYYMHV 1110 1.15E−01 3.93E−033.76E+00 53 53 807B-M0004-H03/M0120-F03 EGLAGVVKRPARFYYYYMDV 8733.26E−03 1.09E+04 2.80E−07 22 17 807B-M0004-H03/M0120-F04EGSARVVIWPAQYYYYYMDF 434 3.53E−03 5.48E+03 4.89E−07 25 34807B-M0004-H03/M0120-F06 EGSARVVKGPARYYYYSMVV 850 2.94E−03 4.44E+034.17E−07 17 27 807B-M0004-H03/M0120-F07 ERSAAVVKWPVRYYYYYMDL 6114.92E−03 1.26E+03 2.41E−06 30 46 807B-M0004-H03/M0120-G02EGWAALVKGPGRYYYYQMYV 749 3.19E−01 4.67E+02 2.80E−05 56 50807B-M0004-H03/M0120-G03 EGSAGVLKGPAKYYYYYMDI 755 1.77E−03 6.24E+032.33E−07 8 12 807B-M0004-H03/M0120-G04 EGSARVVKGPARYYYYYMDV 956 1.81E−031.09E+04 1.64E−07 9 6 807B-M0004-H03/M0120-G10 EGSAGVVKGPARHYYYYMDI 7552.28E−03 9.65E+03 2.30E−07 14 11 807B-M0004-H03/M0120-G12DGLAEEVKGPAQYYYYYIDG 782 7.80E−02 7.72E+04 1.02E−06 51 43807B-M0004-H03/M0120-H03 EAAAGVVKGPARYYYFNMEV 1114 2.27E−03 4.87E+033.99E−07 13 25 807B-M0004-H03/M0120-H04 EGAARAVRRPAGYYHYYMDL 9163.22E−03 1.46E+04 2.16E−07 20 10 807B-M0004-H03/M0120-H06QGWAGVVKWPARYYYYYMDV 3 5.71E−03 ND ND 34 — 807B-M0004-H03/M0120-H10EGLAGVIPRAARYYYYYMDL 800 6.12E−03 1.06E+04 5.45E−07 37 36 *= selectedclones

TABLE 33 Biacore analysis of 807B-M0009-F06, original clone and variantsBiacore koff kon KD Ranking Clone HV-CDR3 nM 1/s 1/Ms nM koff KD807B-M0009-F06/WT VGMSTYAFDI 243 9.70E−02 0.043  2.26 23 25 807B-M0009-VGMSTYGLEI 471 2.79E−02 2.14E+04 1.26E−06 17 18 F06/M0127-A01*807B-M0009- VGMSTYGFDK 418 8.83E−03 5.62E+04 1.56E−07 2 8 F06/M0127-B07807B-M0009- VGMTTYAFDV 390 8.59E−02 0.0434 1.85 20 22 F06/M0127-B08*807B-M0009- VGISTYGFDL 78 1.53E−02 1.74E+05 8.68E−08 10 2 F06/M0127-C10807B-M0009- VGISTYGFDI 324 1.45E−02 5.56E+04 2.57E−07 7 15 F06/M0127-D01*807B-M0009- VGMATYGFDI 346 9.45E−03 6.97E+04 1.34E−07 3 5 F06/M0127-D05807B-M0009- VGISTYGFDV 240 1.49E−02 1.09E+05 1.35E−07 9 6 F06/M0127-E03807B-M0009- VGMSTYGFDI 289 1.05E−02 7.43E+04 1.40E−07 5 7 F06/M0127-E10807B-M0009- VGIPTYSFDI 146 2.24E−02 2.61E+04 8.57E−07 14 17F06/M0127-E11 807B-M0009- VGLATYSFDL 203 1.02E−01 0.0628 1.62 25 20F06/M0127-F07 *807B-M0009- VGMYNYGFDI 221 1.02E−02 1.09E+05 9.38E−08 4 3F06/M0127-F09 807B-M0009- VGMSTYSFDT 271 1.01E−01 0.0453 2.23 24 24F06/M0127-F11 807B-M0009- VGVSTYGFDI 39 1.59E−02 1.47E+05 1.08E−07 11 4F06/M0127-G02 807B-M0009- VGMFTYAFDT 201 7.87E−02 0.0435 1.81 19 21F06/M0127-H04 807B-M0009- VGKSTYGFDI 305 1.79E−02 8.37E+04 2.14E−07 1213 F06/M0127-H05 807B-M0009- VAMTTYGFDL 253 2.41E−02 3.70E+04 6.52E−0716 16 F06/M0128-C01 807B-M0009- VGISSYGFDI 316 1.46E−02 7.76E+041.88E−07 8 11 F06/M0128-D09 807B-M0009- VAMSNYGFDL 159 2.38E−02 1.13E+052.10E−07 15 12 F06/M0128-D12 807B-M0009- VGMTHYAFDI 148 1.98E−027.91E+04 2.50E−07 13 14 F06/M0128-F02 807B-M0009- VGMLTYAFDI 1207.71E−02 0.0765 1.01 18 19 F06/M0128-F08 807B-M0009- VGLPSYSFDI 1158.98E−02 1.24E+05 1.72E−07 22 10 F06/M0128-G09 *807B-M0009- VGMSNYGFDF116 7.78E−03 1.62E+05 4.79E−08 1 1 F06/M0128-H01 807B-M0009- VGMSTYAFDM211 8.98E−02 0.048  1.87 21 23 F06/M0128-H07 807B-M0009- VGLSTYGFDI 2951.31E−02 8.09E+04 1.62E−07 6 9 F06/M0128-H11 *= selected clones

TABLE 34 Biacore screening of 807B-M0079-D10, original clone andvariants koff Clone HV-CDR3 1/s *807B-M0079-D10/WT GLYR 6.02E−03807B-M0079-D10/M0121-A01 GLHR 1.29E−02 807B-M0079-D10/M0121-A02 GLYG1.95E−02 807B-M0079-D10/M0121-A06 GLYH 1.47E−02 807B-M0079-D10/M0121-A08GLHL 2.14E−02 807B-M0079-D10/M0121-A11 GIYR 1.43E−02807B-M0079-D10/M0121-A12 ALAR 1.65E−02 807B-M0079-D10/M0121-B04 GLFR1.07E−02 807B-M0079-D10/M0121-B05 SLYQ 2.31E−02 807B-M0079-D10/M0121-B12GLLL 1.71E−02 807B-M0079-D10/M0121-C01 GQYR 1.93E−02807B-M0079-D10/M0121-C03 GLAR 2.24E−02 807B-M0079-D10/M0121-D01 GLYQ1.34E−02 807B-M0079-D10/M0121-D05 GLYP 5.93E−03 807B-M0079-D10/M0121-D06GMYR 1.62E−02 807B-M0079-D10/M0121-E02 ALYS 5.34E−03807B-M0079-D10/M0121-F02 GLSR 8.02E−03 807B-M0079-D10/M0121-F05 SLYL1.84E−02 807B-M0079-D10/M0121-F06 GLYL 1.11E−02 807B-M0079-D10/M0121-F11GMYV 5.92E−03 807B-M0079-D10/M0121-G03 SLYR 1.22E−02807B-M0079-D10/M0121-G10 ALYR 1.03E−02 807B-M0079-D10/M0121-H04 SLYH2.03E−02 807B-M0079-D10/M0121-H05 GLYY 1.70E−02 807B-M0079-D10/M0122-A01DLYR 1.65E−02 807B-M0079-D10/M0122-B03 TLHR 2.58E−02807B-M0079-D10/M0122-D01 GLHH 2.11E−02 807B-M0079-D10/M0122-D03 GLNR1.29E−02 807B-M0079-D10/M0122-D05 GLSQ 1.43E−02 807B-M0079-D10/M0122-E06GLqR 2.59E−02 807B-M0079-D10/M0122-F09 GLFY 8.76E−03807B-M0079-D10/M0122-F11 GLNL 1.21E−02 807B-M0079-D10/M0122-G07 SLFK2.17E−02 807B-M0079-D10/M0122-G12 ALYW 6.11E−03 807B-M0079-D10/M0122-H11GVYL 1.32E−02 *= selected clones

TABLE 35 Biacore screening of 807A-M0028-B02, original clone andvariants BC koff kon KD Ranking Clone HV-CDR3 nM 1/s 1/Ms nM koff KD*807A-M0028-B02/WT SVLLDY 592 1.48E−02 1.73E+04 869 72 61807A-M0028-B02/M0123-A04 SVQLYP 120 5.05E−02 0.0776 660000000 74 73807A-M0028-B02/M0123-A05 SVLHDK 958 8.85E−03 1.16E+04 768 18 57807A-M0028-B02/M0123-A06 FALLDY 247 7.82E−03 2.20E+04 358 13 28807A-M0028-B02/M0123-A07 SVLFDK 548 6.97E−03 1.96E+04 358 10 27807A-M0028-B02/M0123-A09 TLLLDs 345 1.47E−02 5.85E−03 2530000000 71 74807A-M0028-B02/M0123-A10 GVLLDL 385 9.74E−03 2.13E+04 462 27 40807A-M0028-B02/M0123-A11 SVLFDY 398 1.30E−02 1.29E+04 1020 66 67807A-M0028-B02/M0123-A12 SILFDY 449 1.02E−02 1.36E+04 757 33 56807A-M0028-B02/M0123-B01 SVLLDQ 711 9.49E−03 8.59E+03 1110 25 70807A-M0028-B02/M0123-B03 SNLHDQ 199 1.21E−02 1.12E+04 1080 55 69807A-M0028-B02/M0123-B06 AILLNY 207 7.43E−03 2.89E+04 257 12 15807A-M0028-B02/M0123-B08 AVLLDH 471 1.03E−02 1.38E+04 745 34 54807A-M0028-B02/M0123-B10 AVMHDK 858 3.64E−03 1.80E+04 202 1 8807A-M0028-B02/M0123-C07 SVLFDS 706 1.25E−02 1.32E+04 945 61 63807A-M0028-B02/M0123-C11 GVLLDI 345 9.78E−03 3.11E+04 315 29 20*807A-M0028-B02/M0123-D01 GVLLDK 578 5.62E−03 2.65E+04 212 6 9807A-M0028-B02/M0123-D03 SVLLDN 710 1.12E−02 1.09E+04 1030 45 68807A-M0028-B02/M0123-D04 SVLHDY 592 1.41E−02 1.22E+04 1160 70 71807A-M0028-B02/M0123-D06 SVLFDR 510 9.07E−03 2.13E+04 427 20 35807A-M0028-B02/M0123-D08 SVLLDK 1013 9.26E−03 1.08E+04 862 23 60807A-M0028-B02/M0123-E05 GGLLDY 884 1.06E−02 1.10E+04 976 37 65807A-M0028-B02/M0123-E12 SVMFDY 646 1.13E−02 1.14E+04 1000 46 66807A-M0028-B02/M0123-F01 SILHDY 1049 1.21E−02 7.34E+03 1660 56 72*807A-M0028-B02/M0123-F04 GILHDY 718 4.82E−03 2.07E+04 233 5 13807A-M0028-B02/M0123-F11 SVIFDY 522 1.26E−02 1.72E+04 739 62 53807A-M0028-B02/M0123-F12 SILFDN 737 9.08E−03 1.45E+04 630 21 49807A-M0028-B02/M0123-G02 AILLDY 190 1.55E−02 2.09E+04 747 73 55807A-M0028-B02/M0123-G03 AILLDH 394 1.25E−02 1.76E+04 717 60 52807A-M0028-B02/M0123-G12 SILFDT 628 8.17E−03 2.01E+04 409 15 34807A-M0028-B02/M0123-H02 AVLLDY 276 1.11E−02 2.77E+04 404 42 33807A-M0028-B02/M0123-H09 SVLPDN 685 8.78E−03 2.04E+04 434 16 36807A-M0028-B02/M0123-H10 GILLDK 591 6.36E−03 2.85E+04 224 8 10807A-M0028-B02/M0123-H11 SVLFDN 619 1.15E−02 1.95E+04 596 48 47807A-M0028-B02/M0124-A01 SVLLDS 738 6.01E−03 1.75E+04 344 7 23807A-M0028-B02/M0124-A10 SDLRAE 815 9.75E−03 1.15E+04 855 28 59807A-M0028-B02/M0124-A11 GVLLDY 452 8.85E−03 3.47E+04 257 17 16*807A-M0028-B02/M0124-B02 GVLHDY 649 4.40E−03 2.66E+04 166 3 5807A-M0028-B02/M0124-B03 SVLLDR 201 1.25E−02 4.88E+04 258 59 17807A-M0028-B02/M0124-B08 SILHDK 590 6.37E−03 3.20E+04 200 9 7*807A-M0028-B02/M0124-B11 SILFDK 389 4.49E−03 3.82E+04 118 4 2807A-M0028-B02/M0124-C01 SILLDH 517 1.17E−02 2.00E+04 590 49 46807A-M0028-B02/M0124-C02 SVPIDH 243 1.24E−02 3.63E+04 344 58 24807A-M0028-B02/M0124-C03 PVLLHF 269 8.87E−03 3.87E+04 231 19 11807A-M0028-B02/M0124-C04 GVLLEP 509 9.99E−03 2.05E+04 494 31 41*807A-M0028-B02/M0124-C05 GVLFDN 230 3.93E−03 7.01E+04 56 2 1807A-M0028-B02/M0124-C06 AJLLDK 272 8.08E−03 5.04E+04 162 14 4807A-M0028-B02/M0124-D02 STLLDH 445 1.11E−02 2.07E+04 541 43 43807A-M0028-B02/M0124-D06 SIHLDY 470 1.23E−02 1.40E+04 888 57 62807A-M0028-B02/M0124-D08 SVTLDA 446 1.20E−02 2.23E+04 543 53 45807A-M0028-B02/M0124-D09 SVLHDF 378 1.30E−02 2.87E+04 459 65 39807A-M0028-B02/M0124-D10 SVLHDS 390 1.39E−02 2.22E+04 634 69 51807A-M0028-B02/M0124-D12 GGLLDK 853 7.07E−03 2.16E+04 328 11 22807A-M0028-B02/M0124-E02 AVLLDT 459 1.04E−02 2.74E+04 383 35 30807A-M0028-B02/M0124-E03 AVLHDY 490 1.00E−02 2.21E+04 456 32 38807A-M0028-B02/M0124-E04 SVLHDQ 272 1.21E−02 3.06E+04 399 54 32807A-M0028-B02/M0124-E10 GVLLDN 493 9.68E−03 2.74E+04 357 26 26807A-M0028-B02/M0124-F03 SVLLDH 513 9.24E−03 2.87E+04 325 22 21807A-M0028-B02/M0124-F05 AVLHDS 211 1.19E−02 2.66E+04 451 52 37807A-M0028-B02/M0124-G03 YVHPDY 655 1.32E−02 1.37E+04 971 68 64807A-M0028-B02/M0124-G07 SVLHDH 516 9.81E−03 2.61E+04 380 30 29807A-M0028-B02/M0124-G10 AVLLDN 316 1.08E−02 3.87E+04 282 38 18807A-M0028-B02/M0125-C03 SVLLDR 473 1.18E−02 3.12E+04 385 50 31807A-M0028-B02/M0125-D03 SVLFDY 295 1.11E−02 4.41E+04 254 40 14807A-M0028-B02/M0125-D06 SVHLDY 161 1.12E−02 8.30E+04 136 44 3807A-M0028-B02/M0125-D09 AVLHDS 639 1.06E−02 1.97E+04 542 36 44807A-M0028-B02/M0125-F07 SVLLDQ 607 1.29E−02 2.06E+04 631 64 50807A-M0028-B02/M0125-F11 SVLFDS 621 1.18E−02 2.39E+04 498 51 42807A-M0028-B02/M0125-G02 SVLLDH 511 1.32E−02 2.21E+04 607 67 48807A-M0028-B02/M0126-C09 AVLLDY 329 1.10E−02 3.18E+04 350 39 25807A-M0028-B02/M0126-E03 AVLLDN 257 1.11E−02 3.86E+04 290 41 19807A-M0028-B02/M0126-F08 SILFDY 335 9.35E−03 4.07E+04 232 24 12807A-M0028-B02/M0126-G03 SVLHDN 627 1.28E−02 1.53E+04 846 63 58807A-M0028-B02/M0126-G07 AVLLDH 221 1.15E−02 5.41E+04 194 47 6 *=selected clones BC = Biacore

TABLE 36 Detailed biacore analysis of 807B-M0004-A03, 807B-M0009-F06,807A-M0028-B02 and variants Clone name kon (1/Ms) koff (1/s) KD (nM)CDR3 807B-M004-A03/WT 1.82E+05 2.34E−02 128 SIAAAGTDY807B-M004-A03/M0118-B09 3.12E+05 1.96E−02 63 SIAADRTDY807B-M004-A03/M0117-G01 1.39E+05 7.72E−03 56 SIASARTDS807B-M004-A03/M0117-D03 2.75E+04 8.36E−03 304 SIAAARTDY807B-M004-A03/M0117-F05 1.89E+05 1.05E−02 56 SIASAGTDH807B-M004-A03/M0118-F03 1.35E+05 1.16E−02 86 SIAASRTDY 807B-M0009F06/WT1.31E+05 4.45E−02 340 VGMSTYAFDI 807B-M0009F06-M0128-H01 8.72E+047.99E−03 92 VGMSNYGFDF 807B-M0009F06-M0127-B07 1.00E+05 8.60E−03 86VGMSTYGFDK 807B-M0009F06-M0127-D05 1.02E+05 9.39E−03 92 VGMATYGFDI807B-M0009F06-M0127-F09 1.18E+05 1.10E−02 93 VGMYNYGFDI807B-M0009F06-M0127-C10 1.13E+05 1.62E−02 144 VGISTYGFDL807A-M0028-B02/WT 9.31E+04 1.58E−02 169 SVLLDY 807A-M0028-B02/M0124-C052.76E+04 5.12E−03 185 GVLFDN 807A-M0028-B02/M0124-B02 6.07E+04 6.77E−03112 GVLHDY 807A-M0028-B02/M0124-B11 5.23E+04 5.72E−03 109 SILFDK807A-M0028-B02/M0123-F04 5.07E+04 7.56E−03 149 GILHDY807A-M0028-B02/M0123-D01 9.25E+04 7.85E−03 85 GVLLDK 807B-M0004-H03/WT —— 200 EGSAGVVKGP ARYYYYYMDV 807B-M0004-H03/M0119-B05 — — — EGSSGVVKGPARYYYYYMDA 807B-M0004-H03/M0119-C05 — — — ERSVAVFKAR PRHYYYYMDV807B-M0004-H03/M0119-F04 — — — EGSARVIKGP ARYYYYEMDV807B-M0004-H03/M0119-H09 — — — DGSARVVKGP RRYYYYYIDV807B-M0004-H03/M0120-B05 — — — EGSARVVKGP ARYFYYYMDL

TABLE 37 Immunohistochemistry of clones selected from Biacore screeningIHC Clone 807B-M0004-A03 Original clone +/− 807B-M0004-A03/M0117-D03 +807B-M0004-A03/M0117-F05 + 807B-M0004-A03/M0117-G01 +/−807B-M0004-A03/M0118-B09 +(+) 807B-M0004-A03/M0118-F03 +/− Clone807B-M0004-H03 Original clone +/− 807B-M0004-H03/M0119-B05 ++(+)807B-M0004-H03/M0119-C05 ++(+) 807B-M0004-H03/M0119-F04 ++(+)807B-M0004-H03/M0119-H09 ++(+) 807B-M0004-H03/M0120-B05 ++ Clone807A-M0028-B02 Original clone ++ 807B-M0028-B02/M0123-D01 ++807B-M0028-B02/M0123-F04 ++ 807B-M0028-B02/M0124-B02 ++807B-M0028-B02/M0124-B11 ++ 807B-M0028-B02/M0124-C05 ++ Clone807B-M0009-F06 Original clone +/− 807B-M0009-F06/M0127-B07 +/−807B-M0009-F06/M0127-C10 + 807B-M0009-F06/M0127-D05 −807B-M0009-F06/M0127-F09 +/− 807B-M0009-F06/M0128-H01 +

TABLE 38 Affinity matured clones of 807A-M0028-B02 Initial Name HV-CDR3LV-CDR1 LV-CDR2 LV-CDR3 807A-M0028-B02/M0167-E01 SEQ ID NO: 207 SEQ IDNO: 211 SEQ ID NO: 240 SEQ ID NO: 262 807A-M0028-B02/M0167-E07 SEQ IDNO: 208 SEQ ID NO: 212 SEQ ID NO: 241 SEQ ID NO: 263807A-M0028-B02/M0167-F07 SEQ ID NO: 209 SEQ ID NO: 213 SEQ ID NO: 34 SEQID NO: 35 807A-M0028-B02/M0167-F09 SEQ ID NO: 207 SEQ ID NO: 214 SEQ IDNO: 243 SEQ ID NO: 265 807A-M0028-B02/M0168-B11 SEQ ID NO: 209 SEQ IDNO: 215 SEQ ID NO: 244 SEQ ID NO: 266 807A-M0028-B02/M0168-C08 SEQ IDNO: 209 SEQ ID NO: 216 SEQ ID NO: 245 SEQ ID NO: 267807A-M0028-B02/M0168-D10 SEQ ID NO: 210 SEQ ID NO: 33 SEQ ID NO: 34 SEQID NO: 35 807A-M0028-B02/M0169-D08 SEQ ID NO: 208 SEQ ID NO: 218 SEQ IDNO: 34 SEQ ID NO: 268 807A-M0028-B02/M0169-F03 SEQ ID NO: 209 SEQ ID NO:219 SEQ ID NO: 247 SEQ ID NO: 269 807A-M0028-B02/M0169-H04 SEQ ID NO:208 SEQ ID NO: 220 SEQ ID NO: 248 SEQ ID NO: 270807A-M0028-B02/M0169-H05 SEQ ID NO: 207 SEQ ID NO: 221 SEQ ID NO: 249SEQ ID NO: 271 807A-M0028-B02/M0170-H08 SEQ ID NO: 207 SEQ ID NO: 222SEQ ID NO: 250 SEQ ID NO: 272 807A-M0028-B02/M0171-A08 SEQ ID NO: 210SEQ ID NO: 223 SEQ ID NO: 240 SEQ ID NO: 273 807A-M0028-B02/M0171-A09SEQ ID NO: 209 SEQ ID NO: 224 SEQ ID NO: 251 SEQ ID NO: 274807A-M0028-B02/M0171-A10 SEQ ID NO: 207 SEQ ID NO: 33 SEQ ID NO: 34 SEQID NO: 35 807A-M0028-B02/M0171-C12 SEQ ID NO: 207 SEQ ID NO: 226 SEQ IDNO: 252 SEQ ID NO: 275 807A-M0028-B02/M0171-E03 SEQ ID NO: 209 SEQ IDNO: 218 SEQ ID NO: 34 SEQ ID NO: 268 807A-M0028-B02/M0171-G02 SEQ ID NO:208 SEQ ID NO: 228 SEQ ID NO: 253 SEQ ID NO: 276807A-M0028-B02/M0171-G09 SEQ ID NO: 209 SEQ ID NO: 229 SEQ ID NO: 254SEQ ID NO: 277 807A-M0028-B02/M0172-A05 SEQ ID NO: 209 SEQ ID NO: 230SEQ ID NO: 255 SEQ ID NO: 278 807A-M0028-B02/M0172-A08 SEQ ID NO: 208SEQ ID NO: 231 SEQ ID NO: 256 SEQ ID NO: 279 807A-M0028-B02/M0172-B09SEQ ID NO: 209 SEQ ID NO: 232 SEQ ID NO: 257 SEQ ID NO: 280807A-M0028-B02/M0172-D05 SEQ ID NO: 209 SEQ ID NO: 233 SEQ ID NO: 257SEQ ID NO: 281 807A-M0028-B02/M0172-D09 SEQ ID NO: 207 SEQ ID NO: 234SEQ ID NO: 258 SEQ ID NO: 282 807A-M0028-B02/M0172-E06 SEQ ID NO: 209SEQ ID NO: 235 SEQ ID NO: 251 SEQ ID NO: 283 807A-M0028-B02/M0172-F02SEQ ID NO: 207 SEQ ID NO: 236 SEQ ID NO: 259 SEQ ID NO: 284807A-M0028-B02/M0172-F07 SEQ ID NO: 210 SEQ ID NO: 213 SEQ ID NO: 34 SEQID NO: 35 807A-M0028-B02/M0172-F12 SEQ ID NO: 209 SEQ ID NO: 238 SEQ IDNO: 260 SEQ ID NO: 285 807A-M0028-B02/M0172-G08 SEQ ID NO: 209 SEQ IDNO: 239 SEQ ID NO: 261 SEQ ID NO: 286 Initial Name HV-CDR1 HV-CDR2LV-WholeAA HV-WholeAA 807A-M0028-B02/M0167-E01 SEQ ID NO: 24 SEQ ID NO:25 SEQ ID NO: 287 SEQ ID NO: 316 807A-M0028-B02/M0167-E07 SEQ ID NO: 24SEQ ID NO: 25 SEQ ID NO: 288 SEQ ID NO: 317 807A-M0028-B02/M0167-F07 SEQID NO: 24 SEQ ID NO: 25 SEQ ID NO: 289 SEQ ID NO: 318807A-M0028-B02/M0167-F09 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 290 SEQID NO: 316 807A-M0028-B02/M0168-B11 SEQ ID NO: 24 SEQ ID NO: 25 SEQ IDNO: 291 SEQ ID NO: 318 807A-M0028-B02/M0168-C08 SEQ ID NO: 24 SEQ ID NO:25 SEQ ID NO: 292 SEQ ID NO: 318 807A-M0028-B02/M0168-D10 SEQ ID NO: 24SEQ ID NO: 25 SEQ ID NO: 43 SEQ ID NO: 319 807A-M0028-B02/M0169-D08 SEQID NO: 24 SEQ ID NO: 25 SEQ ID NO: 294 SEQ ID NO: 317807A-M0028-B02/M0169-F03 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 295 SEQID NO: 318 807A-M0028-B02/M0169-H04 SEQ ID NO: 24 SEQ ID NO: 25 SEQ IDNO: 296 SEQ ID NO: 317 807A-M0028-B02/M0169-H05 SEQ ID NO: 24 SEQ ID NO:25 SEQ ID NO: 297 SEQ ID NO: 316 807A-M0028-B02/M0170-H08 SEQ ID NO: 24SEQ ID NO: 25 SEQ ID NO: 298 SEQ ID NO: 316 807A-M0028-B02/M0171-A08 SEQID NO: 24 SEQ ID NO: 25 SEQ ID NO: 299 SEQ ID NO: 319807A-M0028-B02/M0171-A09 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 300 SEQID NO: 318 807A-M0028-B02/M0171-A10 SEQ ID NO: 24 SEQ ID NO: 25 SEQ IDNO: 43 SEQ ID NO: 316 807A-M0028-B02/M0171-C12 SEQ ID NO: 24 SEQ ID NO:25 SEQ ID NO: 302 SEQ ID NO: 316 807A-M0028-B02/M0171-E03 SEQ ID NO: 24SEQ ID NO: 25 SEQ ID NO: 294 SEQ ID NO: 318 807A-M0028-B02/M0171-G02 SEQID NO: 24 SEQ ID NO: 25 SEQ ID NO: 304 SEQ ID NO: 317807A-M0028-B02/M0171-G09 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 305 SEQID NO: 318 807A-M0028-B02/M0172-A05 SEQ ID NO: 24 SEQ ID NO: 25 SEQ IDNO: 306 SEQ ID NO: 318 807A-M0028-B02/M0172-A08 SEQ ID NO: 24 SEQ ID NO:25 SEQ ID NO: 307 SEQ ID NO: 317 807A-M0028-B02/M0172-B09 SEQ ID NO: 24SEQ ID NO: 25 SEQ ID NO: 308 SEQ ID NO: 318 807A-M0028-B02/M0172-D05 SEQID NO: 24 SEQ ID NO: 25 SEQ ID NO: 309 SEQ ID NO: 318807A-M0028-B02/M0172-D09 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 310 SEQID NO: 316 807A-M0028-B02/M0172-E06 SEQ ID NO: 24 SEQ ID NO: 25 SEQ IDNO: 311 SEQ ID NO: 318 807A-M0028-B02/M0172-F02 SEQ ID NO: 24 SEQ ID NO:25 SEQ ID NO: 312 SEQ ID NO: 316 807A-M0028-B02/M0172-F07 SEQ ID NO: 24SEQ ID NO: 25 SEQ ID NO: 289 SEQ ID NO: 319 807A-M0028-B02/M0172-F12 SEQID NO: 24 SEQ ID NO: 25 SEQ ID NO: 314 SEQ ID NO: 318807A-M0028-B02/M0172-G08 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 315 SEQID NO: 318

TABLE 39 Affinity matured clones of 807B-M0004-A03 Initial NameHV-CDR3_1 LV-CDR1 LV-CDR2 LV-CDR3 807B-M0004-A03/M0148-E05 SEQ ID NO:320 SEQ ID NO: 93 SEQ ID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0148-E08SEQ ID NO: 320 SEQ ID NO: 325 SEQ ID NO: 333 SEQ ID NO: 341807B-M0004-A03/M0149-D04 SEQ ID NO: 322 SEQ ID NO: 326 SEQ ID NO: 334SEQ ID NO: 341 807B-M0004-A03/M0149-F02 SEQ ID NO: 322 SEQ ID NO: 93 SEQID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0149-G11 SEQ ID NO: 323 SEQ IDNO: 93 SEQ ID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0149-H07 SEQ ID NO:322 SEQ ID NO: 326 SEQ ID NO: 334 SEQ ID NO: 341807B-M0004-A03/M0149-H09 SEQ ID NO: 320 SEQ ID NO: 327 SEQ ID NO: 333SEQ ID NO: 341 807B-M0004-A03/M0150-A04 SEQ ID NO: 320 SEQ ID NO: 93 SEQID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0150-A07 SEQ ID NO: 321 SEQ IDNO: 93 SEQ ID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0150-A12 SEQ ID NO:320 SEQ ID NO: 93 SEQ ID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0150-D12SEQ ID NO: 320 SEQ ID NO: 325 SEQ ID NO: 333 SEQ ID NO: 341807B-M0004-A03/M0150-E01 SEQ ID NO: 320 SEQ ID NO: 93 SEQ ID NO: 335 SEQID NO: 341 807B-M0004-A03/M0150-E03 SEQ ID NO: 320 SEQ ID NO: 325 SEQ IDNO: 333 SEQ ID NO: 341 807B-M0004-A03/M0150-E04 SEQ ID NO: 320 SEQ IDNO: 327 SEQ ID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0150-E12 SEQ IDNO: 320 SEQ ID NO: 328 SEQ ID NO: 336 SEQ ID NO: 342807B-M0004-A03/M0150-G01 SEQ ID NO: 320 SEQ ID NO: 329 SEQ ID NO: 337SEQ ID NO: 341 807B-M0004-A03/M0151-A06 SEQ ID NO: 320 SEQ ID NO: 330SEQ ID NO: 338 SEQ ID NO: 343 807B-M0004-A03/M0151-B12 SEQ ID NO: 320SEQ ID NO: 325 SEQ ID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0151-C05SEQ ID NO: 320 SEQ ID NO: 328 SEQ ID NO: 336 SEQ ID NO: 342807B-M0004-A03/M0151-D09 SEQ ID NO: 322 SEQ ID NO: 325 SEQ ID NO: 333SEQ ID NO: 341 807B-M0004-A03/M0151-F12 SEQ ID NO: 320 SEQ ID NO: 93 SEQID NO: 333 SEQ ID NO: 341 807B-M0004-A03/M0151-H05 SEQ ID NO: 320 SEQ IDNO: 331 SEQ ID NO: 339 SEQ ID NO: 341 807B-M0004-A03/M0153-D03 SEQ IDNO: 320 SEQ ID NO: 93 SEQ ID NO: 94 SEQ ID NO: 95807B-M0004-A03/M0153-F07 SEQ ID NO: 320 SEQ ID NO: 327 SEQ ID NO: 333SEQ ID NO: 341 Initial Name HV-CDR1 HV-CDR2 LV-WholeAA HV-WholeAA807B-M0004-A03/M0148-E05 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 345 SEQID NO: 369 807B-M0004-A03/M0148-E08 SEQ ID NO: 48 SEQ ID NO: 49 SEQ IDNO: 346 SEQ ID NO: 369 807B-M0004-A03/M0149-D04 SEQ ID NO: 48 SEQ ID NO:49 SEQ ID NO: 347 SEQ ID NO: 370 807B-M0004-A03/M0149-F02 SEQ ID NO: 48SEQ ID NO: 49 SEQ ID NO: 348 SEQ ID NO: 370 807B-M0004-A03/M0149-G11 SEQID NO: 48 SEQ ID NO: 49 SEQ ID NO: 348 SEQ ID NO: 371807B-M0004-A03/M0149-H07 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 350 SEQID NO: 370 807B-M0004-A03/M0149-H09 SEQ ID NO: 48 SEQ ID NO: 49 SEQ IDNO: 351 SEQ ID NO: 369 807B-M0004-A03/M0150-A04 SEQ ID NO: 48 SEQ ID NO:49 SEQ ID NO: 352 SEQ ID NO: 369 807B-M0004-A03/M0150-A07 SEQ ID NO: 48SEQ ID NO: 49 SEQ ID NO: 348 SEQ ID NO: 372 807B-M0004-A03/M0150-A12 SEQID NO: 48 SEQ ID NO: 49 SEQ ID NO: 354 SEQ ID NO: 369807B-M0004-A03/M0150-D12 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 355 SEQID NO: 369 807B-M0004-A03/M0150-E01 SEQ ID NO: 48 SEQ ID NO: 49 SEQ IDNO: 356 SEQ ID NO: 369 807B-M0004-A03/M0150-E03 SEQ ID NO: 48 SEQ ID NO:49 SEQ ID NO: 357 SEQ ID NO: 369 807B-M0004-A03/M0150-E04 SEQ ID NO: 48SEQ ID NO: 49 SEQ ID NO: 358 SEQ ID NO: 369 807B-M0004-A03/M0150-E12 SEQID NO: 48 SEQ ID NO: 49 SEQ ID NO: 359 SEQ ID NO: 369807B-M0004-A03/M0150-G01 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 360 SEQID NO: 369 807B-M0004-A03/M0151-A06 SEQ ID NO: 48 SEQ ID NO: 49 SEQ IDNO: 361 SEQ ID NO: 369 807B-M0004-A03/M0151-B12 SEQ ID NO: 48 SEQ ID NO:49 SEQ ID NO: 362 SEQ ID NO: 369 807B-M0004-A03/M0151-C05 SEQ ID NO: 48SEQ ID NO: 49 SEQ ID NO: 363 SEQ ID NO: 369 807B-M0004-A03/M0151-D09 SEQID NO: 48 SEQ ID NO: 49 SEQ ID NO: 362 SEQ ID NO: 370807B-M0004-A03/M0151-F12 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 348 SEQID NO: 369 807B-M0004-A03/M0151-H05 SEQ ID NO: 48 SEQ ID NO: 49 SEQ IDNO: 366 SEQ ID NO: 369 807B-M0004-A03/M0153-D03 SEQ ID NO: 48 SEQ ID NO:49 SEQ ID NO: 151 SEQ ID NO: 369 807B-M0004-A03/M0153-F07 SEQ ID NO: 48SEQ ID NO: 49 SEQ ID NO: 368 SEQ ID NO: 369

TABLE 40 Affinity matured clones of 807B-M0004-H03 Initial Name HV-CDR3LV-CDR1 LV-CDR2 LV-CDR3 807B-M0004-H03/M0154-C07 SEQ ID NO: 373 SEQ IDNO: 388 SEQ ID NO: 381 SEQ ID NO: 377 807B-M0004-H03/M0154-D08 SEQ IDNO: 373 SEQ ID NO: 389 SEQ ID NO: 382 SEQ ID NO: 378807B-M0004-H03/M0154-G05 SEQ ID NO: 373 SEQ ID NO: 390 SEQ ID NO: 383SEQ ID NO: 379 807B-M0004-H03/M0154-G08 SEQ ID NO: 373 SEQ ID NO: 389SEQ ID NO: 382 SEQ ID NO: 378 807B-M0004-H03/M0154-G11 SEQ ID NO: 374SEQ ID NO: 389 SEQ ID NO: 382 SEQ ID NO: 378 807B-M0004-H03/M0154-H03SEQ ID NO: 375 SEQ ID NO: 389 SEQ ID NO: 382 SEQ ID NO: 378807B-M0004-H03/M0154-H06 SEQ ID NO: 373 SEQ ID NO: 391 SEQ ID NO: 382SEQ ID NO: 378 807B-M0004-H03/M0155-C08 SEQ ID NO: 373 SEQ ID NO: 389SEQ ID NO: 382 SEQ ID NO: 378 807B-M0004-H03/M0155-E10 SEQ ID NO: 376SEQ ID NO: 392 SEQ ID NO: 384 SEQ ID NO: 380 807B-M0004-H03/M0155-F08SEQ ID NO: 373 SEQ ID NO: 389 SEQ ID NO: 382 SEQ ID NO: 378807B-M0004-H03/M0155-H06 SEQ ID NO: 373 SEQ ID NO: 389 SEQ ID NO: 382SEQ ID NO: 378 807B-M0004-H03/M0155-H08 SEQ ID NO: 376 SEQ ID NO: 389SEQ ID NO: 382 SEQ ID NO: 378 807B-M0004-H03/M0156-D01 SEQ ID NO: 373SEQ ID NO: 389 SEQ ID NO: 382 SEQ ID NO: 378 807B-M0004-H03/M0156-G08SEQ ID NO: 373 SEQ ID NO: 389 SEQ ID NO: 382 SEQ ID NO: 378807B-M0004-H03/M0157-A08 SEQ ID NO: 373 SEQ ID NO: 389 SEQ ID NO: 382SEQ ID NO: 378 807B-M0004-H03/M0157-D10 SEQ ID NO: 376 SEQ ID NO: 389SEQ ID NO: 382 SEQ ID NO: 378 807B-M0004-H03/M0157-G08 SEQ ID NO: 373SEQ ID NO: 389 SEQ ID NO: 382 SEQ ID NO: 378 807B-M0004-H03/M0157-G11SEQ ID NO: 375 SEQ ID NO: 393 SEQ ID NO: 385 SEQ ID NO: 378807B-M0004-H03/M0159-A09 SEQ ID NO: 373 SEQ ID NO: 394 SEQ ID NO: 386SEQ ID NO: 378 807B-M0004-H03/M0159-H03 SEQ ID NO: 373 SEQ ID NO: 389SEQ ID NO: 382 SEQ ID NO: 378 807B-M0004-H03/M0159-H07 SEQ ID NO: 373SEQ ID NO: 395 SEQ ID NO: 387 SEQ ID NO: 380 807B-M0004-H03/M0157-F04SEQ ID NO: 68 SEQ ID NO: 396 SEQ ID NO: 382 SEQ ID NO: 378 Initial NameHV-CDR1 HV-CDR2 LV-WholeAA HV-WholeAA 807B-M0004-H03/M0154-C07 SEQ IDNO: 66 SEQ ID NO: 67 SEQ ID NO: 400 SEQ ID NO: 397807B-M0004-H03/M0154-D08 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 401 SEQID NO: 397 807B-M0004-H03/M0154-G05 SEQ ID NO: 66 SEQ ID NO: 67 SEQ IDNO: 402 SEQ ID NO: 397 807B-M0004-H03/M0154-G08 SEQ ID NO: 66 SEQ ID NO:67 SEQ ID NO: 403 SEQ ID NO: 397 807B-M0004-H03/M0154-G11 SEQ ID NO: 66SEQ ID NO: 67 SEQ ID NO: 404 SEQ ID NO: 217 807B-M0004-H03/M0154-H03 SEQID NO: 66 SEQ ID NO: 67 SEQ ID NO: 405 SEQ ID NO: 398807B-M0004-H03/M0154-H06 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 406 SEQID NO: 397 807B-M0004-H03/M0155-C08 SEQ ID NO: 66 SEQ ID NO: 67 SEQ IDNO: 407 SEQ ID NO: 397 807B-M0004-H03/M0155-E10 SEQ ID NO: 66 SEQ ID NO:67 SEQ ID NO: 408 SEQ ID NO: 399 807B-M0004-H03/M0155-F08 SEQ ID NO: 66SEQ ID NO: 67 SEQ ID NO: 409 SEQ ID NO: 397 807B-M0004-H03/M0155-H06 SEQID NO: 66 SEQ ID NO: 67 SEQ ID NO: 410 SEQ ID NO: 397807B-M0004-H03/M0155-H08 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 411 SEQID NO: 399 807B-M0004-H03/M0156-D01 SEQ ID NO: 66 SEQ ID NO: 67 SEQ IDNO: 412 SEQ ID NO: 397 807B-M0004-H03/M0156-G08 SEQ ID NO: 66 SEQ ID NO:67 SEQ ID NO: 413 SEQ ID NO: 397 807B-M0004-H03/M0157-A08 SEQ ID NO: 66SEQ ID NO: 67 SEQ ID NO: 414 SEQ ID NO: 397 807B-M0004-H03/M0157-D10 SEQID NO: 66 SEQ ID NO: 67 SEQ ID NO: 415 SEQ ID NO: 399807B-M0004-H03/M0157-G08 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 416 SEQID NO: 397 807B-M0004-H03/M0157-G11 SEQ ID NO: 66 SEQ ID NO: 67 SEQ IDNO: 417 SEQ ID NO: 398 807B-M0004-H03/M0159-A09 SEQ ID NO: 66 SEQ ID NO:67 SEQ ID NO: 418 SEQ ID NO: 397 807B-M0004-H03/M0159-H03 SEQ ID NO: 66SEQ ID NO: 67 SEQ ID NO: 419 SEQ ID NO: 397 807B-M0004-H03/M0159-H07 SEQID NO: 66 SEQ ID NO: 67 SEQ ID NO: 420 SEQ ID NO: 397807B-M0004-H03/M0157-F04 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 421 SEQID NO: 142

TABLE 41 Affinity matured clones of 807B-M0009-F06 Initial NameHV-CDR3_1 LV-CDR1 LV-CDR2 LV-CDR3 807B-M0009-F06/M0173-F07 SEQ ID NO:485 SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119807B-M0009-F06/M0174-B01 SEQ ID NO: 486 SEQ ID NO: 117 SEQ ID NO: 118SEQ ID NO: 119 807B-M0009-F06/M0174-B06 SEQ ID NO: 487 SEQ ID NO: 117SEQ ID NO: 118 SEQ ID NO: 119 807B-M0009-F06/M0174-B08 SEQ ID NO: 488SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119 807B-M0009-F06/M0175-A07SEQ ID NO: 488 SEQ ID NO: 501 SEQ ID NO: 118 SEQ ID NO: 101807B-M0009-F06/M0175-B01 SEQ ID NO: 485 SEQ ID NO: 502 SEQ ID NO: 497SEQ ID NO: 101 807B-M0009-F06/M0175-B11 SEQ ID NO: 485 SEQ ID NO: 503SEQ ID NO: 498 SEQ ID NO: 119 807B-M0009-F06/M0175-C07 SEQ ID NO: 488SEQ ID NO: 501 SEQ ID NO: 118 SEQ ID NO: 101 807B-M0009-F06/M0175-D04SEQ ID NO: 485 SEQ ID NO: 503 SEQ ID NO: 498 SEQ ID NO: 119807B-M0009-F06/M0175-E04 SEQ ID NO: 485 SEQ ID NO: 503 SEQ ID NO: 118SEQ ID NO: 492 807B-M0009-F06/M0175-E06 SEQ ID NO: 486 SEQ ID NO: 503SEQ ID NO: 118 SEQ ID NO: 119 807B-M0009-F06/M0176-A06 SEQ ID NO: 485SEQ ID NO: 504 SEQ ID NO: 118 SEQ ID NO: 101 807B-M0009-F06/M0176-C04SEQ ID NO: 485 SEQ ID NO: 505 SEQ ID NO: 499 SEQ ID NO: 493807B-M0009-F06/M0176-G02 SEQ ID NO: 485 SEQ ID NO: 225 SEQ ID NO: 118SEQ ID NO: 101 807B-M0009-F06/M0177-E01 SEQ ID NO: 489 SEQ ID NO: 117SEQ ID NO: 118 SEQ ID NO: 119 807B-M0009-F06/M0177-E05 SEQ ID NO: 486SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119 807B-M0009-F06/M0177-E09SEQ ID NO: 488 SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119807B-M0009-F06/M0177-F09 SEQ ID NO: 488 SEQ ID NO: 503 SEQ ID NO: 118SEQ ID NO: 494 807B-M0009-F06/M0177-H02 SEQ ID NO: 488 SEQ ID NO: 503SEQ ID NO: 118 SEQ ID NO: 119 807B-M0009-F06/M0177-H05 SEQ ID NO: 489SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119 807B-M0009-F06/M0177-H06SEQ ID NO: 488 SEQ ID NO: 505 SEQ ID NO: 499 SEQ ID NO: 493807B-M0009-F06/M0177-H07 SEQ ID NO: 488 SEQ ID NO: 506 SEQ ID NO: 118SEQ ID NO: 495 807B-M0009-F06/M0178-A08 SEQ ID NO: 488 SEQ ID NO: 503SEQ ID NO: 118 SEQ ID NO: 496 807B-M0009-F06/M0178-E02 SEQ ID NO: 486SEQ ID NO: 507 SEQ ID NO: 118 SEQ ID NO: 494 807B-M0009-F06/M0178-H09SEQ ID NO: 488 SEQ ID NO: 505 SEQ ID NO: 499 SEQ ID NO: 493 Initial NameHV-CDR1 HV-CDR2 LV-WholeAA HV-WholeAA 807B-M0009-F06/M0173-F07 SEQ IDNO: 72 SEQ ID NO: 73 SEQ ID NO: 508 SEQ ID NO: 500807B-M0009-F06/M0174-B01 SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 508 SEQID NO: 490 807B-M0009-F06/M0174-B06 SEQ ID NO: 72 SEQ ID NO: 73 SEQ IDNO: 508 SEQ ID NO: 491 807B-M0009-F06/M0174-B08 SEQ ID NO: 72 SEQ ID NO:73 SEQ ID NO: 508 SEQ ID NO: 509 807B-M0009-F06/M0175-A07 SEQ ID NO: 72SEQ ID NO: 73 SEQ ID NO: 227 SEQ ID NO: 509 807B-M0009-F06/M0175-B01 SEQID NO: 72 SEQ ID NO: 73 SEQ ID NO: 237 SEQ ID NO: 500807B-M0009-F06/M0175-B11 SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 242 SEQID NO: 500 807B-M0009-F06/M0175-C07 SEQ ID NO: 72 SEQ ID NO: 73 SEQ IDNO: 246 SEQ ID NO: 509 807B-M0009-F06/M0175-D04 SEQ ID NO: 72 SEQ ID NO:73 SEQ ID NO: 264 SEQ ID NO: 500 807B-M0009-F06/M0175-E04 SEQ ID NO: 72SEQ ID NO: 73 SEQ ID NO: 293 SEQ ID NO: 500 807B-M0009-F06/M0175-E06 SEQID NO: 72 SEQ ID NO: 73 SEQ ID NO: 301 SEQ ID NO: 490807B-M0009-F06/M0176-A06 SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 303 SEQID NO: 500 807B-M0009-F06/M0176-C04 SEQ ID NO: 72 SEQ ID NO: 73 SEQ IDNO: 313 SEQ ID NO: 500 807B-M0009-F06/M0176-G02 SEQ ID NO: 72 SEQ ID NO:73 SEQ ID NO: 324 SEQ ID NO: 500 807B-M0009-F06/M0177-E01 SEQ ID NO: 72SEQ ID NO: 73 SEQ ID NO: 332 SEQ ID NO: 510 807B-M0009-F06/M0177-E05 SEQID NO: 72 SEQ ID NO: 73 SEQ ID NO: 340 SEQ ID NO: 490807B-M0009-F06/M0177-E09 SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 344 SEQID NO: 509 807B-M0009-F06/M0177-F09 SEQ ID NO: 72 SEQ ID NO: 73 SEQ IDNO: 349 SEQ ID NO: 509 807B-M0009-F06/M0177-H02 SEQ ID NO: 72 SEQ ID NO:73 SEQ ID NO: 353 SEQ ID NO: 509 807B-M0009-F06/M0177-H05 SEQ ID NO: 72SEQ ID NO: 73 SEQ ID NO: 508 SEQ ID NO: 510 807B-M0009-F06/M0177-H06 SEQID NO: 72 SEQ ID NO: 73 SEQ ID NO: 364 SEQ ID NO: 509807B-M0009-F06/M0177-H07 SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 365 SEQID NO: 509 807B-M0009-F06/M0178-A08 SEQ ID NO: 72 SEQ ID NO: 73 SEQ IDNO: 367 SEQ ID NO: 509 807B-M0009-F06/M0178-E02 SEQ ID NO: 72 SEQ ID NO:73 SEQ ID NO: 439 SEQ ID NO: 490 807B-M0009-F06/M0178-H09 SEQ ID NO: 72SEQ ID NO: 73 SEQ ID NO: 442 SEQ ID NO: 509

TABLE 42 Affinity matured clones of 807B-M0079-D10 Initial Name LV-CDR1LV-CDR2 LV-CDR3 HV-CDR1 807B-M0079-D10/M0160-F02 SEQ ID NO: 422 SEQ IDNO: 434 SEQ ID NO: 444 SEQ ID NO: 78 807B-M0079-D10/M0160-F12 SEQ ID NO:423 SEQ ID NO: 435 SEQ ID NO: 445 SEQ ID NO: 78 807B-M0079-D10/M0161-B04SEQ ID NO: 424 SEQ ID NO: 434 SEQ ID NO: 446 SEQ ID NO: 78807B-M0079-D10/M0161-G03 SEQ ID NO: 423 SEQ ID NO: 435 SEQ ID NO: 445SEQ ID NO: 78 807B-M0079-D10/M0162-A11 SEQ ID NO: 425 SEQ ID NO: 435 SEQID NO: 447 SEQ ID NO: 78 807B-M0079-D10/M0162-D11 SEQ ID NO: 424 SEQ IDNO: 434 SEQ ID NO: 445 SEQ ID NO: 78 807B-M0079-D10/M0162-F04 SEQ ID NO:426 SEQ ID NO: 436 SEQ ID NO: 448 SEQ ID NO: 78 807B-M0079-D10/M0164-B02SEQ ID NO: 423 SEQ ID NO: 435 SEQ ID NO: 445 SEQ ID NO: 78807B-M0079-D10/M0164-B03 SEQ ID NO: 425 SEQ ID NO: 435 SEQ ID NO: 447SEQ ID NO: 78 807B-M0079-D10/M0164-C08 SEQ ID NO: 425 SEQ ID NO: 435 SEQID NO: 447 SEQ ID NO: 78 807B-M0079-D10/M0164-C09 SEQ ID NO: 427 SEQ IDNO: 437 SEQ ID NO: 449 SEQ ID NO: 78 807B-M0079-D10/M0164-C10 SEQ ID NO:428 SEQ ID NO: 438 SEQ ID NO: 450 SEQ ID NO: 78 807B-M0079-D10/M0164-E05SEQ ID NO: 424 SEQ ID NO: 434 SEQ ID NO: 446 SEQ ID NO: 78807B-M0079-D10/M0165-A02 SEQ ID NO: 429 SEQ ID NO: 440 SEQ ID NO: 451SEQ ID NO: 78 807B-M0079-D10/M0165-A06 SEQ ID NO: 430 SEQ ID NO: 441 SEQID NO: 452 SEQ ID NO: 78 807B-M0079-D10/M0165-B07 SEQ ID NO: 423 SEQ IDNO: 435 SEQ ID NO: 445 SEQ ID NO: 78 807B-M0079-D10/M0165-B08 SEQ ID NO:428 SEQ ID NO: 438 SEQ ID NO: 450 SEQ ID NO: 78 807B-M0079-D10/M0165-D03SEQ ID NO: 431 SEQ ID NO: 434 SEQ ID NO: 445 SEQ ID NO: 78807B-M0079-D10/M0165-D05 SEQ ID NO: 428 SEQ ID NO: 438 SEQ ID NO: 450SEQ ID NO: 78 807B-M0079-D10/M0165-D12 SEQ ID NO: 428 SEQ ID NO: 438 SEQID NO: 450 SEQ ID NO: 78 807B-M0079-D10/M0165-E01 SEQ ID NO: 426 SEQ IDNO: 436 SEQ ID NO: 448 SEQ ID NO: 78 807B-M0079-D10/M0166-A08 SEQ ID NO:429 SEQ ID NO: 440 SEQ ID NO: 451 SEQ ID NO: 78 807B-M0079-D10/M0166-A09SEQ ID NO: 424 SEQ ID NO: 434 SEQ ID NO: 446 SEQ ID NO: 78807B-M0079-D10/M0166-B08 SEQ ID NO: 423 SEQ ID NO: 435 SEQ ID NO: 445SEQ ID NO: 78 807B-M0079-D10/M0166-C08 SEQ ID NO: 430 SEQ ID NO: 441 SEQID NO: 452 SEQ ID NO: 78 807B-M0079-D10/M0166-D03 SEQ ID NO: 432 SEQ IDNO: 443 SEQ ID NO: 454 SEQ ID NO: 78 807B-M0079-D10/M0166-F03 SEQ ID NO:423 SEQ ID NO: 435 SEQ ID NO: 445 SEQ ID NO: 78 807B-M0079-D10/M0166-F04SEQ ID NO: 425 SEQ ID NO: 435 SEQ ID NO: 447 SEQ ID NO: 78807B-M0079-D10/M0166-F07 SEQ ID NO: 433 SEQ ID NO: 434 SEQ ID NO: 445SEQ ID NO: 78 807B-M0079-D10/M0166-F08 SEQ ID NO: 424 SEQ ID NO: 434 SEQID NO: 446 SEQ ID NO: 78 Initial Name HV-CDR2 HV-CDR3 LV-WholeAAHV-WholeAA 807B-M0079-D10/M0160-F02 SEQ ID NO: 453 SEQ ID NO: 80 SEQ IDNO: 455 SEQ ID NO: 146 807B-M0079-D10/M0160-F12 SEQ ID NO: 453 SEQ IDNO: 80 SEQ ID NO: 456 SEQ ID NO: 146 807B-M0079-D10/M0161-B04 SEQ ID NO:453 SEQ ID NO: 80 SEQ ID NO: 457 SEQ ID NO: 146 807B-M0079-D10/M0161-G03SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 458 SEQ ID NO: 146807B-M0079-D10/M0162-A11 SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 459 SEQID NO: 146 807B-M0079-D10/M0162-D11 SEQ ID NO: 453 SEQ ID NO: 80 SEQ IDNO: 460 SEQ ID NO: 146 807B-M0079-D10/M0162-F04 SEQ ID NO: 453 SEQ IDNO: 80 SEQ ID NO: 461 SEQ ID NO: 146 807B-M0079-D10/M0164-B02 SEQ ID NO:453 SEQ ID NO: 80 SEQ ID NO: 462 SEQ ID NO: 146 807B-M0079-D10/M0164-B03SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 463 SEQ ID NO: 146807B-M0079-D10/M0164-C08 SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 464 SEQID NO: 146 807B-M0079-D10/M0164-C09 SEQ ID NO: 453 SEQ ID NO: 80 SEQ IDNO: 465 SEQ ID NO: 146 807B-M0079-D10/M0164-C10 SEQ ID NO: 453 SEQ IDNO: 80 SEQ ID NO: 466 SEQ ID NO: 146 807B-M0079-D10/M0164-E05 SEQ ID NO:453 SEQ ID NO: 80 SEQ ID NO: 467 SEQ ID NO: 146 807B-M0079-D10/M0165-A02SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 468 SEQ ID NO: 146807B-M0079-D10/M0165-A06 SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 469 SEQID NO: 146 807B-M0079-D10/M0165-B07 SEQ ID NO: 453 SEQ ID NO: 80 SEQ IDNO: 470 SEQ ID NO: 146 807B-M0079-D10/M0165-B08 SEQ ID NO: 453 SEQ IDNO: 80 SEQ ID NO: 471 SEQ ID NO: 146 807B-M0079-D10/M0165-D03 SEQ ID NO:453 SEQ ID NO: 80 SEQ ID NO: 472 SEQ ID NO: 146 807B-M0079-D10/M0165-D05SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 473 SEQ ID NO: 146807B-M0079-D10/M0165-D12 SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 474 SEQID NO: 146 807B-M0079-D10/M0165-E01 SEQ ID NO: 453 SEQ ID NO: 80 SEQ IDNO: 475 SEQ ID NO: 146 807B-M0079-D10/M0166-A08 SEQ ID NO: 453 SEQ IDNO: 80 SEQ ID NO: 476 SEQ ID NO: 146 807B-M0079-D10/M0166-A09 SEQ ID NO:453 SEQ ID NO: 80 SEQ ID NO: 477 SEQ ID NO: 146 807B-M0079-D10/M0166-B08SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 478 SEQ ID NO: 146807B-M0079-D10/M0166-C08 SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 479 SEQID NO: 146 807B-M0079-D10/M0166-D03 SEQ ID NO: 453 SEQ ID NO: 80 SEQ IDNO: 480 SEQ ID NO: 146 807B-M0079-D10/M0166-F03 SEQ ID NO: 453 SEQ IDNO: 80 SEQ ID NO: 481 SEQ ID NO: 146 807B-M0079-D10/M0166-F04 SEQ ID NO:453 SEQ ID NO: 80 SEQ ID NO: 482 SEQ ID NO: 146 807B-M0079-D10/M0166-F07SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 483 SEQ ID NO: 146807B-M0079-D10/M0166-F08 SEQ ID NO: 453 SEQ ID NO: 80 SEQ ID NO: 484 SEQID NO: 146

TABLE 43 Light chain sequences of antibodies selected by VH-CDR3 spikingand light chain shuffling Initial Name LV-FR1 LV-CDR1 LV-FR2 LV-CDR2807B-M0004-A03/M0149-D04 QSVLTQSPSASGT SGSNSNVGTKTVN WYQVLPGTAPKLLIYSNTQRPS PGQRVTISC 807B-M0004-A03/M0149-F02 QSALTQPPSASGT SGSSSNIGSNTVNWYQQLPGTAPKLLIY SNNQRPS PGQRVTISC 807B-M0004-A03/M0150-A07 QSALTQPPSASGTSGSSSNIGSNTVN WYQQLPGTAPKLLIY SNNQRPS PGQRVTISC 807B-M0004-A03/M0150-E03QYELTQPPSASGT SGSSSNIGINTVN WYQQLPGTAPKLLIY SNNQRPS PGQRVTISC807B-M0004-A03/M0151-D09 QSALTQPPSASGT SGSSSNIGINTVN WYQQLPGTAPKLLIYSNNQRPS PGQRVTISC 807A-M0028-B02/M0169-F03 QDIQMTQSPSSLS QASQDISNYLNWYQQKPGKAPQRLIY GASTVQS ASVGDRVTITC 807A-M0028-B02/M0171-E03QDIQMTQSPSSLS RTSQDIGNHLA WYQQKPGKAPQRLIR EASILQS ASVGDRVTITC807A-M0028-B02/M0171-G02 QDIQMTQSPSSLS RASQGITNYLA WFQQKPGKAPKSLMYGAYKLQY ASVGDTVTITC 807A-M0028-B02/M0172-F07 QDIQMTQSPSSLS RTSQGIRNHLGWFQQKPGKAPQRLIR EASILQS ASVGDRVTITC 807B-M0004-H03/M0154-H06QDIQMTQSPSSLS RASRGVSTSLN WYQIKPEKAPKLLIY AASSLQS ASVGDRVTITC807B-M0004-H03/M0159-A09 QDIQMTQSPSSLS RASQSIRSYLN WFQQKPGKAPKLLIYAASTLQS ASVGDRVTITC 807A-M0028-B02/M0168-D10 QDIQMTQSPSSLS RTSQDIRNHLGWFQQKPGKAPQRLIR EASILQS ASVGDRVTITC 807B-M0004-A03/M0149-G11QSALTQPPSASGT SGSSSNIGSNTVN WYQQLPGTAPKLLIY SNNQRPS PGQRVTISC InitialName LV-FR3 LV-CDR3 LV-FR4 807B-M0004-A03/M0149-D04GVPDRFSGSKSGTSASLAISG AAWDDSLNGPV FGGGTRVTVL LQSEDEADYYC807B-M0004-A03/M0149-F02 GVPDRFSGSKSGTSASLAISG AAWDDSLNGPV FGGGTKLTVLLQSEDEADYYC 807B-M0004-A03/M0150-A07 GVPDRFSGSKSGTSASLAISG AAWDDSLNGPVFGGGTKLTVL LQSEDEADYYC 807B-M0004-A03/M0150-E03 GVPDRFSGSKSGTSASLAISGAAWDDSLNGPV FGGGTKLTVL LQSEDEADYYC 807B-M0004-A03/M0151-D09GVPDRFSGSKSGTSASLAISG AAWDDSLNGPV FGGGTKLTVL LQSEDEADYYC807A-M0028-B02/M0169-F03 GVPSRFSGSGSGTEFTLTISS QQYKTYPFT FGQGTRLDIKLQPDDFATYYC 807A-M0028-B02/M0171-E03 GVPSTFSGSGSGTEFTLTISS QQYDAFPFTFGQGTKLEIK LQPEDFASYYC 807A-M0028-B02/M0171-G02 GVPTKFSGSGSGTDFTLTIRSLQYQTYPFT FGPGTKVDLK LQPEDFATYYC 807A-M0028-B02/M0172-F07GVPSTFSGSGSGTEFTLTISS LQYDSFPYT FGQGTKLEIK LQPEDFATYYC807B-M0004-H03/M0154-H06 GVPSRFSGSGSGTDFTLAITS QQSYSTPRT FGPGTKVEIKLQPEDFATYYC 807B-M0004-H03/M0159-A09 GVPSRFSGSGSGTDFTLTISS QQSYSTPRTFGQGTKLEIK LQPEDFATYYC 807A-M0028-B02/M0168-D10 GVPSTFYGSGYGREFTLTISSLQYDSFPYT FGQGTKLEIK LQPEDFATYYC 807B-M0004-A03/M0149-G11GVPDRFSGSKSGTSASLAISG AAWDDSLNGPV FGGGTKLTVL LQSEDEADYYC

TABLE 44 Heavy chain sequences of antibodies selected by VH-CDR3 spikingand light chain shuffling HV- Initial Name HV-FR1 CDR1 HV-FR2 HV-CDR2807B-M0004-A03/M0149-D04 EVQLLESGGGLVQPGGSLRL RYLMM WVRQAPGKGLEWVSVISPSGGRTWYADSVKG SCAASGFTFS 807B-M0004-A03/M0149-F02EVQLLESGGGLVQPGGSLRL RYLMM WVRQAPGKGLEWVS VISPSGGRTWYADSVKG SCAASGFTFS807B-M0004-A03/M0150-A07 EVQLLESGGGLVQPGGSLRL RYLMM WVRQAPGKGLEWVSVISPSGGRTWYADSVKG SCAASGFTFS 807B-M0004-A03/M0150-E03EVQLLESGGGLVQPGGSLRL RYLMM WVRQAPGKGLEWVS VISPSGGRTWYADSVKG SCAASGFTFS807B-M0004-A03/M0151-D09 EVQLLESGGGLVQPGGSLRL RYLMM WVRQAPGKGLEWVSVISPSGGRTWYADSVKG SCAASGFTFS 807A-M0028-B02/M0169-F03EVQLLESGGGLVQPGGSLRL MYMMD WVRQAPGKGLEWVS SIWPSGGQTWYADSVKG SCAASGFTFS807A-M0028-B02/M0171-E03 EVQLLESGGGLVQPGGSLRL MYMMD WVRQAPGKGLEWVSSIWPSGGQTWYADSVKG SCAASGFTFS 807A-M0028-B02/M0171-G02EVQLLESGGGLVQPGGSLRL MYMMD WVRQAPGKGLEWVS SIWPSGGQTWYADSVKG SCAASGFTFS807A-M0028-B02/M0172-F07 EVQLLESGGGLVQPGGSLRL MYMMD WVRQAPGKGLEWVSSIWPSGGQTWYADSVKG SCAASGFTFS 807B-M0004-H03/M0154-H06EVQLLESGGGLVQPGGSLRL SYPMV WVRQAPGKGLEWVS GIWSSGGLTYYADSVKG SCAASGFTFS807B-M0004-H03/M0159-A09 EVQLLESGGGLVQPGGSLRL SYPMV WVRQAPGKGLEWVSGIWSSGGLTYYADSVKG SCAASGFTFS 807A-M0028-B02/M0168-D10EVQLLESGGGLVQPGGSLRL MYMMD WVRQAPGKGLEWVS SIWPSGGQTWYADSVKG SCAASGFTFS807B-M0004-A03/M0149-G11 EVQLLESGGGLVQPGGSLRL RYLMM WVRQAPGKGLEWVSVISPSGGRTWYADSVKG SCAASGFTFS Initial Name HV-FR3 HV-CDR3 HV-FR4807B-M0004-A03/M0149-D04 RFTISRDNSKNTLYLQMNSLRAEDTA SIASAGTDHWGQGTLVTVSS VYYCVR 807B-M0004-A03/M0149-F02 RFTISRDNSKNTLYLQMNSLRAEDTASIASAGTDH WGQGTLVTVSS VYYCVR 807B-M0004-A03/M0150-A07RFTISRDNSKNTLYLQMNSLRAEDTA SIAASRTDY WGQGTLVTVSS VYYCVR807B-M0004-A03/M0150-E03 RFTISRDNSKNTLYLQMNSLRAEDTA SIAADRTDYWGQGTLVTVSS VYYCVR 807B-M0004-A03/M0151-D09 RFTISRDNSKNTLYLQMNSLRAEDTASIASAGTDH WGQGTLVTVSS VYYCVR 807A-M0028-B02/M0169-F03RFTISRDNSKNTLYLQMNSLRAEDTA GVLLDK WGQGTLVTVSS VYYCAR807A-M0028-B02/M0171-E03 RFTISRDNSKNTLYLQMNSLRAEDTA GVLLDK WGQGTLVTVSSVYYCAR 807A-M0028-B02/M0171-G02 RFTISRDNSKNTLYLQMNSLRAEDTA GILHDYWGQGTLVTVSS VYYCAR 807A-M0028-B02/M0172-F07 RFTISRDNSKNTLYLQMNSLRAEDTAGVLFDN WGQGTLVTVSS VYYCAR 807B-M0004-H03/M0154-H06RFTISRDNSKNTLYLQMNSLRAEDTA ERSVAVFKARPRHYYYY WGKGTTVTVSS VYYCAR MDV807B-M0004-H03/M0159-A09 RFTISRDNSKNTLYLQMNSLRAEDTA ERSVAVFKARPRHYYYYWGKGTTVTVSS VYYCAR MDV 807A-M0028-B02/M0168-D10RFTISRDNSKNTLYLQMNSLRAEDTA GVLFDN WGQGTLVTVSS VYYCAR807B-M0004-A03/M0149-G11 RFTISRDNSKNTLYLQMNSLRAEDTA SIASARTDSWGQGTLVTVSS VYYCVR

1. An isolated human, humanised or chimeric antibody or antibodyfragment, which antibody or fragment: (i) binds to a polypeptide havingthe amino acid sequence shown in SEQ ID NO: 1 of the C-terminal domainof Apolipoprotein E (ApoE-CTD) or the amino acid sequence of a partthereof; (ii) binds to human plaques; and (iii) comprises: (a) a heavychain CDR3 region comprising the sequence shown in SEQ ID NO: 26, SEQ IDNO: 20, SEQ ID NO: 23, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209,SEQ ID NO: 210, or SEQ ID NO: 512; (b) a heavy chain CDR2 regioncomprising the sequence shown in SEQ ID NO: 25; (c) a heavy chain CDR1region comprising the sequence shown in SEQ ID NO: 24; (d) a light chainCDR3 region comprising the sequence shown in SEQ ID NO: 35; (e) a lightchain CDR2 region comprising the sequence shown in SEQ ID NO: 34; and(f) a light chain CDR1 region comprising the sequence shown in SEQ IDNO:
 33. 2. An antibody or antibody fragment according to claim 1,wherein said heavy chain CDR3 region comprises an affinity maturedvariant of SEQ ID NO: 26 having the sequence shown in SEQ ID NO:
 512. 3.An antibody or antibody fragment according to claim 1, wherein saidheavy chain CDR3 region comprises an affinity matured variant of SEQ IDNO: 26 having the sequence shown in SEQ ID NO:
 20. 4. An antibody orantibody fragment according to claim 1 wherein said heavy chain CDR3region comprises the sequence shown in SEQ ID NO:
 23. 5. An antibody orantibody fragment according to claim 2 wherein said heavy chain CDR3region comprises an affinity matured variant of SEQ ID NO: 26 having thesequence shown in SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209 or SEQID NO:
 210. 6. An antibody or antibody fragment according to claim 5,wherein said heavy chain CDR3 region comprises an affinity maturedvariant of SEQ ID NO: 26 having the sequence shown in SEQ ID NO: 207,SEQ ID NO: 208 or SEQ ID NO:
 209. 7. An antibody or antibody fragmentaccording to claim 1, wherein said polypeptide having the amino acidsequence of a part of SEQ ID NO: 1 comprises the sequence shown in SEQID NO:
 5. 8. An antibody or antibody fragment according to claim 1,wherein said ApoE-CTD polypeptide is a recombinant polypeptide.
 9. Anantibody or antibody fragment according to claim 8 wherein saidrecombinant polypeptide is biotinylated.
 10. An antibody or antibodyfragment according to claim 1, which binds to said plaques in thepresence of VLDL.
 11. An antibody or antibody fragment according toclaim 1, wherein said VLDL is present in human plasma.
 12. An antibodyor antibody fragment according to claim 11, which binds to the plaquesin the presence of 25% plasma.
 13. An antibody or antibody fragmentaccording to claim 12, which binds to the plagues in the presence offrom 25% to 50% plasma.
 14. An antibody or antibody fragment accordingto claim 13, which binds to the plaques in the presence of 50% plasma.15. An isolated antibody or antibody fragment which comprises: (a) theheavy chain sequence shown in SEQ ID NO: 40 and the light chain sequenceshown in SEQ ID NO: 518 and/or 519 or (b) the heavy chain sequence shownin SEQ ID NO: 40 and the light chain sequence shown in SEQ ID NO: 520and/or
 521. 16. An isolated antibody or antibody fragment whichcomprises: (a) the heavy chain CDR1 sequence shown in SEQ ID NO: 24, theheavy chain CDR2 sequence shown in SEQ ID NO: 25 and the heavy chainCDR3 sequence shown in any one of SEQ ID NOS: 207, 209 and 210; and (b)the light chain ODR1, CDR2 and CDR3 sequences shown in SEQ ID NOS: 33,34 and 35, SEQ ID NOS: 219, 247 and 269, SEQ ID NOS: 226, 252 and 275 orSEQ ID NOS: 218, 34 and
 268. 17. An antibody or antibody fragmentaccording to claim 16 wherein the heavy chain CDR3 region comprises thesequence shown in SEQ ID NO: 210 and the light chain comprises thesequences shown in SEQ ID NOS: 33, 34 and 35, the heavy chain CDR3region comprises the sequence shown in SEQ ID NO: 209 and the lightchain comprises the sequences shown in SEQ ID NOS: 219, 247 and 269 orSEQ ID NOS: 218, 34 and 268, or the heavy chain CDR3 region comprisesthe sequence shown in SEQ ID NO: 207 and the light chain comprises thesequence shown in SEQ ID NOS: 226, 252 and
 275. 18. An antibody orantibody fragment according to claim 16, wherein the heavy chaincomprises the sequence shown in any one of SEQ ID NO: 317, 318 or 319.19. An antibody or antibody fragment according to claim 16, wherein thelight chain comprises the sequence shown in SEQ ID NO: 43, 295, 294 or304.
 20. An antibody or antibody fragment according to claim 1, whereinsaid antibody is an lgG.
 21. An antibody or antibody fragment accordingto claim 1, wherein said antibody fragment is a Fab fragment or scFv.22. An antibody or antibody fragment according to claim 1, which is amonoclonal antibody.
 23. An antibody or antibody fragment according toclaim 1, which is a humanised antibody.
 24. An antibody or antibodyfragment according to claim 1, which is chimeric.
 25. A pharmaceuticalcomposition comprising an antibody or antibody fragment according toclaim 1 and a pharmaceutically acceptable carrier or diluent.
 26. A kitfor detecting ApoE-CTD, which kit comprises an antibody or antibodyfragment according to claim 1 and means for detecting said an antibodyor antibody fragment.
 27. An antibody or antibody fragment according toclaim 1, which is a human antibody.